1Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908; and 2Huntsman Cancer Institute and Department of Biology, University of Utah, Salt Lake City, Utah 84112
Submitted 31 December 2002 ; accepted in final form 16 May 2003
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ABSTRACT |
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LIM protein; dense plaque; Rho-kinase; nuclear transport; cell migration
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MATERIALS AND METHODS |
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Western blot analysis. Tissue homogenates were submitted to
SDS-PAGE, and the proteins were transferred to polyvinylidene difluoride
(PVDF) membranes. The membranes were blocked with either 10% donkey serum (for
donkey anti-goat antibodies) or 5% nonfat dry milk in phosphate-buffered
saline containing 0.05% Tween 20 (PBS-T) for 1 h and then incubated with the
primary antibody for2hat room temperature or overnight at 4°C. The blots
were washed in PBS-T, incubated in secondary antibody for 1 h at room
temperature, and revealed by enhanced chemiluminescence (ECL; PerkinElmer Life
Sciences, Boston, MA). The following dilutions of primary antibodies were
used: goat polyclonal anti-Vav2, 1:1,000 (Santa Cruz Biotechnology, Santa
Cruz, CA); rabbit anti-zyxin B71 and rabbit anti-zyxin B38 antibodies,
1:5,000; mouse monoclonal anti-smooth muscle -actin, 1:100,000 (Sigma);
mouse monoclonal anti-
actin, 1:5,000 (Sigma); mouse anti-vinculin,
1:10,000 (Sigma); mouse anti-glyceraldehyde 3-phosphate dehydrogenase,
1:10,000 (GAPDH; Chemicon, Temecula, CA); rabbit anti-guanine nucleotide
dissociation inhibitor (GDI), 1:5,000 (Santa Cruz Biotechnology); and rabbit
serum response factor (SRF) anti-serum, 1:500 (Santa Cruz Biotechnology). A
custom affinity-purified rabbit polyclonal antibody specific for LPP, used at
the dilution of 1:20,000, was made using a KLH-conjugated synthetic peptide
(GQQGHPNTWKREPGY; 318332) derived from the human LPP sequence
(University of Virginia Biomolecular Research Facility, Charlottesville,
VA).
Two-dimensional gel electrophoresis and protein sequencing. Protein samples were run on 2-D gel electrophoresis using a mini-Protean II 2-D gel apparatus (Bio-Rad). Proteins (100 µg) were loaded on acrylamide gels prepared with an ampholyte mixture of pH 310 and were separated by nonequilibrium pH gel electrophoresis (NEPHGE) at 400 V constant voltage for 6 h. After migration, the gels were equilibrated for 15 min in Laemmli buffer (11). The tube gels were loaded on 8% SDS-acrylamide gels for migration in the second dimension. After migration, the gels were either silver-stained or transferred on PVDF membranes for Western blotting. For protein sequencing, the spots extracted from the silver-stained gels were destained overnight in 50% methanol, and the gel pieces were dehydrated in acetonitrile and rehydrated in 10 mM DTT and 0.1 M ammonium bicarbonate and reduced at room temperature for 30 min. The proteins were digested with trypsin overnight at 37°C, and the peptides formed were extracted from the gel and microsequenced by LC-mass spectrometry and Edman degradation at the Biomolecular Research Facility of the University of Virginia.
Expression and purification of recombinant full-length LPP. The
LPP cDNA was cloned from a human kidney cDNA library (Ambion, Austin, TX)
using primers 5'-CGT ATC GAA TTC TCT CAC CCA TCT TGG CTG CCA
CCC and 3'-TTC CTC GAG CTA AAG ATC AGT GCT CGC CTT GGC GGT
C. The primers incorporated EcoR1 and XhoI restriction
sites for in-frame cloning into pHIS-Parallel, which provides an
amino-terminal (His)6 affinity tag
(30). The resultant plasmid
was used to transform Escherichia coli strain BL21(DE3) Codon Plus
RIL (Stratagene, La Jolla, CA). The bacteria were grown in minimal defined
medium supplemented with 1% glucose
(38) to an optical density of
0.7 at 650 nm. The expression of recombinant proteins was induced by the
addition of 1 mM isopropyl thiogalactoside for 2 h. The cells were opened by
three passages in a French pressure cell at 18,000 lbs./in.2 in a
buffer containing 50 mM Tris · HCl, pH 8.0, 5 mM EDTA, 10% glycerol, 1
mM AEBSF, and 1 mM benzamidine and then centrifuged at 14,000 g for
45 min. The recombinant proteins were resuspended from the inclusion bodies in
the pellet in a solution containing 8 M urea and 50 mM
2--mercaptoethanol. After dilution of the sample to achieve a
2-
-mercaptoethanol concentration of <10 mM, the recombinant protein
was purified on a nickel-NTA Superflow column (Qiagen, Valencia, CA) as
described by the manufacturer for purification of proteins in denaturing
conditions. The purity of the protein was calculated to be 91% of total
proteins.
Plasmids expressing human LPP and transfections. The fusion construct of LPP and enhanced green fluorescent protein (EGFP) was prepared by inserting the full sequence of LPP into the EcoRI/KpnI restriction sites of the expression vector pEGFP-C2 (BD Clontech). Human iliac vein smooth muscle cells (HIVS-125) were transfected using the optimized protocol for electroporation of human aortic smooth muscle cells with the use of the Nucleofector apparatus (Amaxa, Cologne, Germany). Seventy percent confluent cells (below 10th passage) were trypsinized, centrifuged, and resuspended in 100 µl of solution (AoSMC Nucleofector solution) and then electroporated in the presence of 5 µg of plasmid by using the program U25. The cells were seeded on gelatin-coated plastic dished and used after 24 h for migration assay.
Quantitation of LPP in smooth muscle. Rabbit portal vein, ileum longitudinal smooth muscle, and abdominal aortic segments were dissected, freed of connective tissue, weighed, and homogenized in SDS buffer (20 µl/mg tissue wet wt) as described in Preparation of tissue extracts. Serial dilutions of each homogenate were used for quantitation over the linear range of the antibody signal on Western blots, utilizing as standards known quantities of purified recombinant LPP transferred to the same PVDF membranes. The protein signals were quantitated by densitometry using an Epson Expression 1,600 densitometer and analyzed with NIH Image 1.62 software.
Cultured cells and dissociated cells. Smooth muscle cells from rat thoracic aorta (gift of Dr. G. K. Owens, University of Virginia) were isolated and cultured as previously described (32). The cells, used at the 10th-26th passage, were seeded on coverslips and cultured until subconfluent in Dulbecco's modified Eagle's medium/F-12 (Life Technologies) containing 10% fetal calf serum (Hyclone, Logan, UT), 100 U/ml penicillin (Life Technologies), and 100 µg/ml streptomycin (Life Technologies) and supplemented with 0.68 mM L-glutamine (Sigma). HIVS-125 cells were purchased from the American Type Culture Collection (ATCC) and cultivated in Ham's F-12K medium with 10% fetal bovine serum and 2 mM L-glutamine and complemented with 50 µg/ml heparin and 0.03 mg/ml endothelial cell growth supplement (ECGS). ECGS and heparin have been shown to help maintain the proliferation of HIVS-125 cells up to 1012 passages, a stage at which the cells' doubling time starts to increase noticeably (37).
Guinea pig bladder smooth muscle cells were isolated as previously described (31), except that papain (1 mg/ml; Sigma) and type II collagenase (1 mg/ml; Sigma) were used to disperse the cells. The cell suspensions were washed by centrifugation to remove the enzymes, and then calcium was reintroduced by the addition of CaCl2 in four incremental steps to recover a concentration of 1.2 mM CaCl2. The freshly dispersed guinea pig bladder cells were spun onto slides for 3 min at 900 g using a cytospin centrifuge (Thermo Shandon, Pittsburgh, CA) and immediately fixed for immunofluorescence staining.
Indirect immunofluorescence. The guinea pig tissues were prepared
for cryosectioning as previously described
(13). Tissues were fixed for 1
h at room temperature or overnight at 4°C in 3% paraformaldehyde in 10 mM
PBS, pH 7.4, and cryoprotected in 5% sucrose-PBS for 60 min followed by 15%
sucrose-PBS for another 60 min before being frozen in liquid
N2-cooled Freon-22. Sections (78 µm) were cut from
tissues embedded in Tissue-Tek OCT compound (Miles, Elkhart, IN),
permeabilized for 10 min with 0.3% Triton X-100 in PBS, rinsed, and then
blocked for 1 h in 1% BSA-PBS at room temperature. Cryosections were labeled
with the rabbit polyclonal LPP (318332) antibody (dilution 1:2,000),
anti-vinculin (1:500, Sigma), or anti--actinin (1:300; Sigma). After
three washings in PBS, a rhodamine red-X-conjugated goat anti-rabbit antibody
or Alexa488-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR)
was used at 1:1,000 dilution for a 1-h incubation. Slides were mounted using
Aqua Poly/Mount (Polysciences, Warrington, PA) as anti-fading agent. The
confocal images were obtained with a Bio-Rad MRC-1000 laser scanning confocal
imaging system equipped with a krypton-argon laser and Zeiss Axiovert 35
microscope.
Rat aortic cultured cells and freshly dispersed guinea pig bladder cells
were fixed with 3% paraformaldehyde for 10 min, rinsed several times with PBS,
and permeabilized with 0.3% Triton X-100 solution in PBS for 5 min. After they
were rinsed, the cells were blocked for 1 h in PBS containing 1% BSA and 1 mM
sodium azide and then incubated with an anti-LPP primary antibody (1:200;
immunoGlobe, Himmelstadt, Germany) for rat cells or the LPP (318332)
rabbit polyclonal antibody (1:2,000) for guinea-pig cells, anti-vinculin
antibody (1:500, Sigma), anti-p120 catenin (1:200, Transduction Labs,
Lexington, KY), or anti--actinin (1:300, Sigma) diluted in blocking
solution for 2 h at room temperature or overnight at 4°C. The secondary
antibodies used were a rhodamine red-X-conjugated goat anti-rabbit IgG (1:200;
Molecular Probes), a rhodamine (TRITC)-F(ab')2 goat
anti-mouse IgG (1:250; Jackson ImmunoResearch, West Grove, PA), or a FITC-goat
anti-rabbit IgG (1:200; Molecular Probes) diluted in blocking solution. Some
cells were labeled for 1 h with rhodamine-phalloidin (1:1,000; Molecular
Probes). The cells were washed four times for 5 min with PBS before being
mounted with Aqua Poly/Mount.
Cell migration assay. The migration assay was performed using Transwell culture inserts with polyethylene terephthalate membranes (8-µm pores, Falcon; Becton Dickinson Labware, Franklin Lakes, NJ). Twenty-four hours posttransfection with LPP or control vectors, the human iliac vein smooth muscle cells were harvested with trypsin-EDTA, resuspended in serum-free medium, counted, and distributed at a density of 5 x 104 cells in the inserts or seeded in separate wells for Western blot analysis or detection of EGFP fluorescence. Each determination was performed in triplicate. The cells were allowed to settle for 1 h before the addition of EGF (10 nM) in the lower chamber and then allowed to migrate to the underside of the insert's membrane for 4 h at 37°C, 5% CO2. At the end of the experiment, the cells were fixed in methanol. Cells on the upper surface of the membrane were mechanically removed with a cotton swab. Cells that migrated to the lower surface of the membrane were stained with crystal violet and counted from three different fields with a x20 objective.
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RESULTS |
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LPP is highly expressed in smooth muscle tissues. Western blotting
with the specific LPP (318332) antibody revealed that LPP was present
at high levels in uterus, corpus cavernosum, portal vein, stomach, aorta,
bladder, and ileum tissues, which are composed of a large proportion of smooth
muscle cells (Fig.
2A). The same tissues also gave a strong signal when
blotted for the smooth muscle-specific isoform of -actin, whereas
-actin was found to be widely distributed. The absence of
-actin
signal in skeletal muscle suggests that the antibody used did not recognize
the
-actin isoform expressed in these cells, because Coomassie staining
of the gels showed a similar amount of total protein, as well as strong actin
and myosin staining. In given that lung, a faint band for LPP was detected but
likely represents LPP from smooth muscle in blood vessels and bronchi in this
tissue, because LPP was detected in pulmonary vessels and bronchi, whereas
parenchyma, when dissected from the most peripheral part of the lung, did not
show a strong signal (Fig.
2B). In contrast to smooth muscle, in tissues such as
skeletal muscle, heart, liver, kidney, brain, and spleen, LPP was detected
only with longer exposures of the PVDF membranes to film, and these low
signals were also associated with stronger labeling of the smooth muscle
-actin (data not shown). Comparison of the LPP contents of bladder and
heart, after concentration of the protein, showed a much higher level of LPP
in bladder than heart (Fig.
2C).
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The amounts of LPP in rabbit ileum longitudinal smooth muscle, abdominal
aorta, and portal vein were determined by quantitative Western blotting, using
serial dilutions of known concentrations of purified recombinant human
(His)6-LPP protein to generate a standard curve. Both the Vav2
antibody and the rabbit polyclonal anti-LPP (318332) recognized the
recombinant (His)6-LPP. Preliminary experiments verified that LPP
of human and rabbit origin were recognized with similar affinity by our LPP
antibody (data not shown). The amounts of LPP in tissues, interpolated from
the standard, were 0.89 ng/µg tissue proteins in ileum longitudinal muscle,
1.09 ng/µg in aortic smooth muscle, and 0.45 ng/µg in portal vein. The
estimated cellular concentrations, calculated on the assumption that 80% of
tissue weight is water and the recombinant (His)6-LPP molecular
weight is 66.7 kDa, were 3.4, 4.1, and 1.7 µM, for ileum, aorta, and portal
vein, respectively. In addition, tissue LPP was 8-fold greater than in
the smooth muscle HIVS-125 cells and 100-fold greater than LPP content in
nonmuscle cells such as NIH/3T3 cells or human umbilical vein endothelial
cells (HUVEC; data not shown).
Cellular distribution of LPP. Aortic tissue homogenates were separated into pellet and supernatant fractions under varying NaCl concentrations (Fig. 3). At salt concentrations >150 mM, LPP moved from the 800 g pellet to the supernatant fraction. The cytosolic protein Rho-GDI was only found in the supernatant fraction as expected, indicative of little supernatant contamination of the pellet fraction, whereas the SRF, located in nuclei, was not extracted from the pellet by 300 mM salt concentrations. Vinculin and actin were found in both fractions, and their distribution was independent of salt concentration. This experiment shows that LPP binds weakly to a structure or cytoskeletal element sedimenting in the 800 g pellet in tissue homogenate and is dissociated by salt concentrations >200 mM, presumably indicative of electro-static binding to a pellet protein.
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Localization of LPP by immunofluorescence in tissues and isolated
cells. Immunofluorescence microscopy on guinea pig ileum, heart, kidney,
and bladder sections was performed using our custom-made anti-LPP antibody
(318332), which showed monospecific reactivity in immunoblots for these
tissues. Guinea pig tissues were used because the secondary antibody gave a
much lower background staining than in rabbit tissues used for the biochemical
experiments. As shown in Fig.
4A, LPP distribution in ileum is mainly associated with
the longitudinal and circular layers of smooth muscle cells. In the mucosal
layer, low-grade staining was observed, which was partly due to nonspecific
staining caused by the secondary antibody. Further experiments are necessary
to clarify whether the slight labeling associated with the epithelial layer
lining the villi is a specific signal for LPP. In bladder sections, LPP
staining was strongly associated with smooth muscle cells
(Fig. 4D) and, as in
heart or kidney, was present in blood vessels
(Fig. 4, BD).
In transverse sections of bladder smooth muscle bundles, confocal microscopy
revealed discontinuous staining localized at the periphery of the cells
(Fig. 5A). The
cytosolic compartments showed weak homogenous staining. Immunofluorescence
patterns of vinculin and LPP in cross sections of muscle cells in the tissue
showed strong overlap (data not shown). In freshly isolated bladder cells,
surface scanning with confocal microscopy showed discontinuous LPP staining in
a riblike pattern (Fig. 5, B and
C) that resembles the localization of vinculin at dense
plaques or dense bodies. Different optical sections of the cell also showed
that LPP is mostly associated with the cell membrane, because intracellular
space presented much weaker staining (Fig.
5D). Dual staining of LPP and vinculin also showed very
strong overlap in the cellular distribution of these proteins in both tissue
sections (Fig. 6,
AC) and isolated cells
(Fig. 6, GI).
In these images, vinculin staining was strictly associated with the plasma
membrane. The peripheral pattern of LPP staining was significantly different
than that of -actinin, which showed extensive intracellular dotlike
labeling (Fig. 6, DF and
JL).
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Our anti-LPP (318332) antibody has low affinity for cells of rat
origin; therefore, we used an anti-LPP antibody directed against the
NH2-terminal portion (1109) of the protein (immunoGlobe) for
labeling of the cultured rat aortic cells. Staining of LPP in cultured smooth
muscle cells showed no colocalization with the cadherin-associated armadillo
protein p120 catenin (Fig. 7,
GI), which is concentrated at cell-cell adhesions,
or with -actinin, known to associate with stress fibers
(Fig. 7, JL).
In contrast, LPP was detected at the end of stress fibers at the level of
focal adhesions (Fig. 7,
DF), showing complete overlap with vinculin
labeling (Fig. 7,
AC).
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Rho-kinase inhibition increases leptomycin B-induced LPP nuclear localization. To determine the effects of Rho-kinase inhibition on LPP localization by immunofluorescence, we treated the cells with 10 µM of the Rho-kinase inhibitor Y-27632 for 2 h before stimulation with leptomycin B or vehicle for an additional 3 h. Rat aortic cells treated with Y-27632 (10 µM) exhibited typical changes in cell shape, with disappearance of vinculin-stained focal adhesions and stress fibers (data not shown), as well as the appearance of cell processes (Fig. 8E). Concomitantly with the decrease in vinculin immunofluorescence signal, staining for LPP was decreased at focal adhesions and slightly increased in the cytosol (Fig. 8E). Although some nuclei were occasionally stained with anti-LPP antibody in control cells, as shown in Fig. 8, A and B, incubation of the cells for 3 h with the irreversible inhibitor of nuclear export, leptomycin B, increased the number of cells containing stained nuclei (Fig. 8F), as observed previously for zyxin (17). Nuclei of cells pretreated for 2 h with Y-27632 before the addition of leptomycin B showed much higher intensity and frequency of LPP labeling than those in cells treated with leptomycin B alone (Fig. 8G). The total cellular amount of LPP after treatment with Y-27632, leptomycin B, or both combined was not altered over the time course of the experiments (Fig. 8C).
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Effects of LPP overexpression on smooth muscle cells migration.
HIVS-125 cells are human iliac smooth muscle vein cells that are reported to
express smooth muscle specific markers
(37). We used these cells for
the migration experiments because their phenotype can be modulated according
to the culture conditions. The amount of LPP protein in HIVS-125 cultured
cells was eightfold lower than in adult rabbit aorta when GAPDH was taken as
reference (data not shown). In the presence of ECGS in the medium (still in
the presence of 10% serum), the cells showed a reduction of LPP total content
(40%) as well as a reduction in cellular smooth muscle
-actin, as
previously described (37).
Transfection of human iliac vein smooth muscle cells with EGFP-human LPP
fusion protein or EGFP alone was performed by electroporation using an
optimized protocol for human smooth muscle aortic cells (Amaxa). The levels of
transfection for HIVS-125 cells were similar for both vectors and were
commonly found between 50 and 80% of total cells as assessed by counting of
fluorescent cells. The overall expression of LPP was increased 10-fold
compared with the initial endogenous LPP levels
(Fig. 9F). In
addition, the EGFP fusion protein did not interfere with LPP binding to its
target, because most of the fluorescence was located on the focal adhesions
(Fig. 9G). The EGFP-
or EGFP-LPP-transfected cells were seeded in Transwells, and the number of
cells that migrated in the lower chamber in response to EGF, used as
chemoattractant, were counted. Figure 9,
AD, shows representative fields of the membrane
after cell fixation and staining. Fluorescence imaging of nonstained cells on
the lower surface of the membrane showed that the migrating cells expressed
the EGFP constructs (data not shown). The percentage of cells stimulated with
EGF that migrated to the lower surface of the membrane was significantly
higher (2.5-fold) compared with unstimulated controls (vehicle). In addition,
in the presence of EGF, the total number of migrated cells transfected with
EGFP-LPP was 100% higher than the number of cells transfected with EGFP
alone.
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DISCUSSION |
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The LPP gene was initially discovered as a gene whose 3' region was
translocated and fused to the high mobility gene 1C (HMG1C) with a
particularly high frequency in lipoma tumors
(19,
21). The translocation was
later found in other tumor cell types
(27). Truncation of the HMG1C
protein, rather than the fusion of the three LIM domains of the LPP sequence
to HMG1C, is thought to be responsible for the tumor phenotype
(1,
6,
27), and no definite function
in tumors has yet been attributed to LPP. Of specific interest is the fact
that we found the LPP protein highly expressed only in smooth muscle. LPP was
detectable in the vascular system as well as other smooth muscle cell types,
including intestine, stomach, uterus, bladder, esophagus, and corpus
cavernosum smooth muscle. Western blots with a smooth muscle-specific
-actin antibody showed a tight correlation between the presence of LPP
and this smooth muscle marker. The differences observed between the expression
of the protein reported in our study and the pattern of expression of LPP mRNA
in Northern blots (21) may be
explained by the presence of blood vessels in organs such as heart, lung, or
kidney, as well as the possible lack of correlation between mRNA expression
and protein content. Quantitation of LPP in aorta, portal vein, and ileum
longitudinal muscle showed that cellular amounts of LPP are
0.51
ng/µg protein, which translates into a cellular concentration of
24 µM. This concentration is consistent with the possibility of
LPP having a significant role in modulating the structure of the cytoskeleton
and its relation to the plasma membrane. LPP was previously found in cultured
cell lines such as human foreskin fibroblasts (HFF), colon carcinoma cells
(Caco2), or keratinocytes
(20); therefore, LPP
expression is not strictly limited to smooth muscle. However, besides its high
content in smooth muscle tissue (100-fold greater than in nonmuscle cultured
cells), LPP content was also eightfold higher in cultured human iliac vein
smooth muscle cells (HIVS-125), than in NIH/3T3 fibroblasts or human
endothelial cells (HUVECs; data not shown).
The subcellular distribution of LPP was determined in bladder tissue and freshly isolated bladder smooth muscle cells. In cross sections of bladder tissue, the labeling showed punctate staining on the periphery of the smooth muscle cells as previously reported for vinculin (2, 7). Vinculin has been shown to be a component of plasma membrane-associated dense bodies (2) and can form riblike staining on the surface of certain cells such as guinea pig taenia coli (33). Electron micrographs show dense bodies in longitudinal sections of vas deferens muscle with bundles of actin filaments of opposite polarity emerging from either side similar to the Z bands of striated bundles, forming chains of minisarcomeres throughout the cytoplasm and converging toward membrane dense bodies (3). Immunofluorescence microscopy with LPP and vinculin antibodies showed colocalization of these proteins (Fig. 5) and their exclusion from the cytosolic compartment, suggesting that, like vinculin, LPP is largely localized to membrane dense bodies, points of fixation of actin filaments to the membrane. Interestingly, the LIM protein zyxin has also been reported to be present in chicken gizzard dense plaques (4).
LPP colocalized with vinculin at focal adhesions in cultured aortic smooth
muscle cells (Fig. 5) and did
not colocalize with -actinin or the cadherin-binding armadillo p120
catenin (24,
25).
-Actinin is
associated with actin filaments in stress fibers but can also bind numerous
cytoskeleton- and membrane-bound proteins, participating in the anchorage of
F-actin to the membrane. Contrary to the reported interaction of LPP with
-actinin (14), we found
very little overlap between LPP and
-actinin distribution in tissues or
isolated smooth muscle cells. In smooth muscle,
-actinin is associated
with both peripheral and cytoplasmic dense bodies, whereas vinculin associates
only with peripheral dense bodies
(3). Our results indicate that
LPP is also a predominantly peripheral protein. The light cytosolic
immunostaining of LPP did not show a pattern significantly overlapping the
-actinin staining. In addition, the fluorescence of overexpressed
EGFP-LPP in the smooth muscle cells, as well as other cell types, is not
strikingly associated with actin or actin-binding proteins along the stress
fibers (present study and Ref.
18). In cultured cells, focal
adhesion plaques are sites of recruitment of integrins, mediating cell
adhesion and supporting cell motility
(12). Dissociation of focal
adhesions by inhibitors of the RhoA/Rho-kinase pathway is accompanied by a
decrease in cell motility (35,
36), and the dissociation of
LPP from focal adhesions in the presence of a Rho-kinase inhibitor (present
study) suggests its role as a regulated component of focal complexes. Whereas
LPP anchors to these structures through its group of three LIM domains
(18), the binding partners
responsible for focal adhesion targeting are as yet unknown. Our results show
that overexpression of LPP, in vascular smooth muscle cells, enhanced
EGF-stimulated migration. The possible binding of LPP to VASP through its
NH2-terminal proline-rich consensus sequence
(20) may account for LPP
effects, because VASP is known to regulate the actin polymerization processes
required for cell motility (see review, Ref.
23). It will be of interest to
determine the behavior and function of LPP in smooth muscle cells migrating in
injured blood vessels and also to determine whether LPP has a signaling
function as part of macromolecular complexes at smooth muscle dense plaques
(8).
An inhibitor of CRM1-mediated nuclear export, leptomycin B, caused accumulation of LPP in the nucleus, as previously reported for LPP (20), zyxin (17), and TRIP6 (40), suggesting the existence of a normally active LPP shuttling mechanism between nucleus and cytoplasm. Our finding of nuclear LPP in untreated vascular smooth muscle cells in culture suggests that this is a normal physiological process modulated by drugs and, perhaps, other agents. The mechanism of nuclear import of LPP is unknown. The effects of leptomycin B were greatly potentiated by a specific Rho-kinase inhibitor, Y-27632 (Fig. 8). Y-27632 changed cell morphology (39), a previously described effect of RhoA inhibition (26), resulting in a decrease in cell size, the formation of long processes, and inhibition of cell migration (34). The Y-27632-induced disassembly of stress fibers and focal adhesions was accompanied by LPP staining of the cytosolic compartment and at the termini of the few remaining stress fibers, indicating that Rho-kinase can modulate LPP localization. Leptomycin B alone did not cause dissociation of focal adhesions, suggesting that inhibition of the CRM1 nuclear exporter is sufficient to increase nuclear localization of LPP. The continuous disassembly/reassembly of adhesion proteins in migrating cells (41) suggests that these proteins are periodically disassociated and physiologically available for nuclear transport, as also indicated by the presence of LPP in the nuclei of some untreated cells (present study). Given the relatively large size of LPP, it is unlikely to be driven into the nucleus by mass action and free diffusion. Y-27632 may act by either removing a constitutive Rho-kinase-mediated brake on nuclear import or inhibiting a redundant export mechanism, parallel to CRM1, through which some LPP could still exit the nucleus even in the presence of leptomycin B. The dissolution of focal adhesions containing LPP by Y-27632 suggests that peripheral localization of LPP depends on phosphorylation of LPP itself, or of a partner protein, by a Rho-kinase dependent mechanism. Considering the functional consequences of nuclear accumulation of LPP, we note that Y-27632 inhibits transcriptional regulation of smooth muscle-specific promoters, an effect tentatively ascribed to nuclear accumulation of G-actin (16). Our present finding of nuclear LPP localization modulated by a Rho-kinase inhibitor raises the possibility that LPP participates in modulating transcriptional regulation of cytoskeletal proteins by Rho-kinase.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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