Developmentally regulated expression of calponin isoforms and
the effect of h2-calponin on cell proliferation
M. Moazzem
Hossain1,
Daw-Yang
Hwang1,
Qi-Quan
Huang1,
Yasuharu
Sasaki2, and
Jian-Ping
Jin1
1 Department of Physiology and Biophysics, Case
Western Reserve University School of Medicine, Cleveland, Ohio
44106-4970; and 2 Department of Pharmacology,
Kitasato University, 5-9-1, Sirogane, Minatoku 108-8641, Japan
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ABSTRACT |
h2-Calponin is found in
both smooth muscle and nonmuscle cells, and its function remains to be
established. Western blots with specific monoclonal antibodies detected
significant expression of h2-calponin in the growing embryonic stomach
and urinary bladder and the early pregnant uterus. Although the
expression of h1-calponin is upregulated in the stomach and bladder
during postnatal development, the expression of h2-calponin is
decreased to low levels in quiescent smooth muscle cells. To
investigate a hypothesis that h2-calponin regulates the function of the
actin cytoskeleton during cytokinesis, a smooth muscle-originated cell
line (SM3) lacking calponin was transfected to express either sense or
antisense h2-calponin cDNA and the effects on the rates of cell
proliferation were examined. Both stable and transient sense
cDNA-transfected cells had a significantly decreased proliferation rate
compared with the antisense cDNA-transfected or nontransfected cells.
Immunofluorescence microscopy showed that the force-expressed
h2-calponin was associated with actin-tropomyosin microfilaments. The
number of binuclear cells was significantly greater in the sense
cDNA-transfected culture, in which h2-calponin was concentrated in a
nuclear ring structure formed by actin filaments. The results suggest
that h2-calponin may regulate cytokinesis by inhibiting the activity of
the actin cytoskeleton.
smooth muscle development; cytokinesis; tropomyosin; actin
cytoskeleton; monoclonal antibody; transfective expression
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INTRODUCTION |
CALPONIN IS A
FAMILY of actin filament-associated proteins. Three isoforms of
calponin, h1 (9, 33, 43), h2 (42), and acidic
(1, 45), have been identified. Isoelectric points (pIs) of
these three calponin isoforms show that h1-calponin is basic (pI = 8.5-9.2), h2-calponin is neutral (pI = 7.2-7.6), and acidic calponin is, as expected, acidic (pI = 5.5-5.8). The
extensively investigated chicken gizzard calponin is equivalent to
mammalian h1-calponin, the major calponin found in smooth muscle cells. The smooth muscle calponin has been shown to inhibit actin-activated myosin ATPase, which has led to a model in which it functions as a
modulator of smooth muscle contractility (30, 41, 48).
Actin-myosin interaction-based motility is essential for cytokinesis, a
process in which the membrane and cytoplasm of a cell are partitioned
through the ingression of a cleavage furrow to form two daughter cells
(8, 12, 14). Cleavage furrow ingression requires a
contractile cortical ring of actin and myosin (28, 38,
39); thus the activity of the actin cytoskeleton has an effect
on cell division (15). Actin-myosin interaction also powers cell proliferation by driving cytoplasmic streaming, which may
contribute to the division of the cytosolic components of the cell
during cytokinesis. Accordingly, through the inhibition of actin-myosin
interaction, calponin may play a role in regulating the functions of
the actin cytoskeleton, such as coordinating changes in cell shape and
intracellular molecular trafficking, both of which are critical events
in cytokinesis (15). Indeed, forced expression of chicken
gizzard calponin in cultured smooth muscle cells and fibroblasts showed
an inhibition of cell proliferation (19). Therefore,
calponin, through its regulation of actin-myosin interaction and
possibly actin filament stability, may function as a controlling factor
for cytokinesis and the rate of cell proliferation.
The conservation in primary structure between the h1 and h2 isoforms of
calponin indicates that they most likely function through similar
molecular mechanisms. However, the extensive sequence diversity and
differences in physical properties between the two isoforms suggest
that they have adapted to divergent biological activities
(5). Because expression of h1-calponin in smooth muscle is
upregulated during differentiation and development (7, 11, 13,
32, 46), it may have a role in the functional maturation of
smooth muscle myofilaments. On the other hand, the tissue distribution,
developmental regulation, and functional significance of h2-calponin
are not well understood. Whereas h1-calponin may play a modulator role
in tuning smooth muscle contractility as previously discussed
(48), the potential role of h2-calponin in regulating the
function of the actin cytoskeleton needs to be investigated.
In the present study, we investigated the expression of h2-calponin
during development and its effect on cell proliferation. Using an
immortalized vascular smooth muscle cell line (SM3; Ref. 37) with no endogenous calponin, we examined the effects
of transfective expression of h2-calponin on the function of the actin
cytoskeleton and cell proliferation. We found that this forced
expression of h2-calponin significantly decreased the rate of cell
proliferation. The expressed h2-calponin associated with actin-tropomyosin thin filaments and caused an increased number of
binuclear cells in which h2-calponin was concentrated in a nuclear ring
structure formed by actin filaments. The data suggest that h2-calponin
suppresses cytokinesis by inhibiting the activity of actin
cytoskeleton. Further supported by its regulated expression in uterus
smooth muscle during pregnancy, h2-calponin may play a role in
modulating cell proliferation during tissue growth and remodeling.
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MATERIALS AND METHODS |
Specific antibodies against calponin isoforms.
Two monoclonal antibodies (MAbs) raised against chicken gizzard
calponin (CP1 and CP3; Ref. 21), which react to mammalian h1-calponin but not h2-calponin (Fig. 1),
were used in the present study to detect the expression of mouse
h1-calponin. A polyclonal antiserum (RAH2) raised against mouse
h2-calponin with a weak cross-reaction to h1-calponin (Fig. 1) was
first used to examine the expression of h2-calponin in cell cultures.

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Fig. 1.
Specific antibodies against h1- and h2-calponins.
A: cloned mouse h1- and h2-calponins expressed in and
purified from Escherichia coli culture were analyzed by
Western blotting with the CP1 and CP3 monoclonal antibodies (MAbs) and
the RAH2 antiserum and alkaline phosphatase-labeled anti-mouse IgG or
anti-rabbit IgG second antibodies, respectively. SDS-polyacrylamide gel
electrophoresis (PAGE) resolved the size difference between the 2 calponin isoforms, and the immunoblots demonstrate that the CP1 and CP3
MAbs are specific to h1-calponin, whereas the anti-h2-calponin RAH2
antiserum showed the expected weaker cross-reaction to h1-calponin.
B: Western blots demonstrate that MAb CP21 is specific to
h2-calponin, whereas MAb CP23 has a weak cross-reaction to h1-calponin
and MAb CP11 recognizes both h1- and h2-calponins.
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To develop MAbs specific to h2-calponin, mouse h2-calponin
(32) was used to immunize 8-wk-old female BALB/c mice in a
short-term immunization protocol (47). The mice were
injected intraperitoneally with 50 µg of purified h2-calponin antigen
in 100 µl of phosphate-buffered saline (PBS) mixed with an equal
volume of Freund's complete adjuvant. Ten days later, one mouse was
intraperitoneally boosted two times with 200 µg each of the antigen
in 200 µl of PBS without adjuvant on two consecutive days. Two days
after the last boost, spleen cells were harvested from the immunized
mouse and fused with SP2/0-Ag14 mouse myeloma cells (American Type
Culture Collection) with 50% polyethylene glycol 3400 containing 7.5%
dimethyl sulfoxide (DMSO) as described previously
(21). Hybridoma colonies were selected by HAT (0.1 mM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine) in
Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum (FBS) and screened by indirect enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase-labeled goat anti-mouse total
immunoglobulin (Sigma) second antibody. The anti-h2-calponin antibody-secreting hybridomas were subcloned three or four times by a
limiting dilution method with young BALB/c mouse spleen cells as
feeders to establish stable cell lines. The hybridoma lines were then
cultured to produce high-titer supernatant and introduced into
2,6,10,14-tetramethyl pentadecane (pristane; Sigma)-primed peritoneal
cavity of BALB/c mice to produce MAb-enriched ascites fluids
(21). The specificity of the MAbs was verified by Western blot analysis (Fig. 1). The anti-h2-calponin MAb CP21, showing no
cross-reaction to h1-calponin, was used in Western blots to examine the
expression of h2-calponin.
Construction of expression vectors.
The coding region of mouse h2-calponin cDNA (32) was first
subcloned into the pBluescript KS(
) plasmid for the isolation of an
EcoRV-SmaI restriction fragment with two blunt
ends. The cDNA coding template was then cloned into the
EcoRV site of the G418-resistant pcDNA3 eukaryotic
expression vector (Invitrogen) downstream of the cytomegalovirus (CMV)
promoter in sense or antisense orientations. The recombinant pcDNA3
plasmids encoding sense and antisense h2-calponin cDNA were identified
by ApaI and PstI restriction enzyme mapping and
verified by DNA sequencing with the dideoxy chain termination method as
described previously (18). The sense expression construct
encodes a nonfusion full-length mouse h2-calponin protein for authentic
functional characterization, and the antisense construct provided a
transfection control in the present study. The recombinant pcDNA3
plasmid DNA was prepared from transformed JM109 Escherichia
coli in large quantities with an alkaline lysis method followed by
ion-exchange chromatography.
SM3 cell culture and transfection.
SM3 is an immortalized cell line derived from rabbit aortic smooth
muscle cells (37). The SM3 cells were cultured in DMEM containing 10% FBS, penicillin (100 µg/ml), and streptomycin (100 µg/ml) at 37°C in 5% CO2.
Transfection of SM3 cells was carried out with the
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomal transfection reagent (Boehringer Mannheim) following the manufacturer's
instructions. SM3 cells were seeded on Corning 10-cm culture dishes at
2 × 106 cells per dish and grown until the monolayer
cells reached 60-80% confluence. Twenty micrograms of the
recombinant supercoil plasmid DNA in 50 µl of TE buffer (10 mM
Tris · HCl, pH 8.0, and 1 mM EDTA) was mixed with
100 µl of DOTAP in 20 mM HEPES buffer (pH 7.3) and incubated at room
temperature for 20 min. The DOTAP-DNA mixture was then gently mixed
with 5 ml of DMEM containing 10% FBS and added to the culture dish to
replace the old medium. The SM3 cell monolayer was incubated with the
DOTAP-DNA medium for 18 h at 37°C in 5% CO2 before
the change to fresh medium.
In transient transfection experiments, the cell cultures were continued
in DMEM containing 10% FBS, penicillin (100 µg/ml), and streptomycin
(100 µg/ml) at 37°C in 5% CO2 and the cells were harvested at a series of time points for characterization. In the
establishment of stable transfection of SM3 cells, the transfected cells were cultured in DMEM containing 10% FBS plus G418 (500 µg/ml;
ICN Biomedical). Results from testing the tolerance of nontransfected
SM3 cells to G418 showed that this cell line is highly sensitive to
G418. In culture medium containing 20 µg/ml G418, all cells died
after 9 days. The recombinant pcDNA3-transfected SM3 cell colonies
resistant to G418 were individually picked up from the culture dish by
trypsin digestion in small cylinders greased to the dish. The cells
were expanded for extracting DNA to verify the transfection by PCR as
described previously (18). The expression of h2-calponin
in the sense cDNA-transfected cells was examined on total cellular
protein extract by Western blotting with the RAH2 antibody. The SM3
cell lines stable-transfected with the sense or antisense h2-calponin
cDNA expression constructs were expanded and stored in DMEM containing
35% FBS and 10% DMSO in liquid nitrogen for later phenotype characterization.
SDS-polyacrylamide gel electrophoresis and Western blotting.
To examine h2-calponin expression in the transfected SM3 cells, as well
as h1- and h2-calponins in smooth muscle tissues from New Zealand White
rabbits and C57B6 mice, SDS-polyacrylamide gel electrophoresis (PAGE)
and Western blotting were carried out as described previously
(47).
The smooth muscle layer of the tissue samples were homogenized in SDS
gel electrophoresis sample buffer (50 mM
Tris · HCl, pH 6.8, 1% SDS, 140 mM
-mercaptoethanol, 0.1% bromphenol blue, 10% glycerol) with a
Polytron-type high-speed tissue homogenizer (PRO Scientific, Monroe,
CT) to extract total cellular proteins. The h2-calponin sense and
antisense cDNA-transfected SM3 cells were suspended from the culture
dishes with Versene solution (in mM: 0.537 EDTA, 136.8 NaCl, 2.68 KCl,
8.1 Na2HPO4, 1.47 KH2PO4, pH 7.2) and washed three times with
PBS, pH 7.2. The elimination of trypsin digestion from the collection
of cells avoided enzymatic degradation of the cellular proteins. SDS
gel sample buffer was added to lyse the cells, and the total protein
was extracted by vortexing.
After heating at 80°C for 5 min and clarification by centrifugation,
the tissue or cell samples were applied on a 12% gel with an
acrylamide-to-bisacrylamide ratio of 29:1 prepared in the Laemmli
discontinuous buffer system. After electrophoresis, the SDS gels were
fixed and stained with Coomassie blue R250 to confirm sample integrity
and optimize the amount of loading. The loading amounts of different
samples were normalized by the area and intensity of the actin band.
Protein bands in duplicate gels were electrophoretically transferred to
a nitrocellulose membrane with a Bio-Rad semidry transfer apparatus at
4-5 mA/cm2 for 30 min. The blotted membranes were
blocked with 1% bovine serum albumin (BSA) in Tris-buffered saline
(TBS; in mM: 150 NaCl and 50 Tris · HCl, pH 7.5)
before the incubation with anti-calponin primary antibodies. After
washes with TBS containing 0.05% Tween 20, the membranes were further
incubated with alkaline phosphatase-labeled anti-rabbit IgG or
anti-mouse IgG second antibody (Sigma). After final washes of the
Western blot membrane, the expression of calponin isoforms was revealed
by incubation in 5-bromo-4-chloro-3-indolyl phosphate and nitro blue
tetrazolium chromogenic substrates. Purified mouse h2- and h1-calponin
expressed in E. coli (32) were used as positive
controls in the SDS-PAGE and Western blot experiments.
Densitometry analysis of the Western blots was done on images scanned
at 600 dpi, and the NIH Image program (version 1.61) was used to
quantify the levels of calponin isoform expression. The calponin bands
detected in Western blots were normalized against the actin band in the
parallel SDS gel to correct for the minor differences in the total
protein concentration among the samples.
Measurement of cell numbers in culture.
A number of different methods are currently in use for direct or
indirect measurements of cell numbers in culture to monitor cell
proliferation. Crystal violet staining is a rapid and sensitive method
for cell number measurement in monolayer cultures (10, 23). In this method, cell nuclei are stained with the crystal violet dye and the excess dye is washed out before the crystal violet
absorbed to the cell nuclei is extracted for optical density (OD)
measurements, which reflect the number of cells in the sample.
To investigate the effects of h2-calponin on cell proliferation, we
have adopted the crystal violet method to measure the number of SM3
cells in culture. Cells in 96-well culture plates containing 200 µl
of medium/well were fixed by adding 20 µl of 11% glutaraldehyde
solution. After gentle shaking at room temperature for 15 min, the
plates were washed three times with double-distilled water and air
dried. The plates were then stained with 100 µl of 0.1% crystal
violet (Sigma) in 20 mM 2-(N-morpholino)ethanesulfonic acid
(MES) buffer (pH 6.0). After gentle shaking at room temperature for 20 min, excess dye was removed by extensive washing with double-distilled water and the plates were air dried before extraction of the bound dye
with 100 µl of 10% acetic acid. Optical density of the dye extracts
was measured at 595 nm (OD595) with an automated microtiter plate reader (Benchmark; Bio-Rad Labs).
To evaluate the accuracy of this method for measuring different types
of cell cultures, we first tested the procedure on uniformly seeded
SP2/0Ag14 mouse myeloma cells. The cells were cultured in DMEM
containing 10% FBS, penicillin (100 µg/ml), and streptomycin (100 µg/ml) at 37°C in 5% CO2. Cells in log phase growth
were harvested by gentle blowing with a Pasteur pipette. The cell
numbers were counted in a hemacytometer before seeding in 96-well
culture plates in DMEM containing 10% FBS. Six hours after seeding,
the cells were fixed and processed for crystal violet staining as described above. The results, shown in Fig.
2, A and B,
demonstrate a very good linear relationship between the
OD595nm values of crystal violet nuclear staining and the
wide range of cell numbers seeded in the culture plate (2 × 102-8 × 104 cells/well).

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Fig. 2.
Measurement of cell numbers by crystal violet staining.
A and B: SP2/0Ag14 mouse myeloma cells were
seeded in 96-well tissue culture plates in DMEM containing 10% FBS at
low and high series of numbers, respectively, and stained with the
crystal violet method after incubation at 37°C in 5% CO2
for 6 h. C and D: SM3 cells were seeded in
96-well culture plates at low and high series of numbers, respectively,
and cultured and stained by crystal violet as in A and
B. Results plotted from means ± SD of quartet experiments
demonstrate excellent linear relationships between the optical density
values at 595 nm (OD595) and the cell numbers for both cell
types over a wide range of cell numbers.
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SM3 cells growing as an attached monolayer were then examined. The cell
numbers were counted for seeding in 96-well culture plates. After
incubation for 6 h in DMEM containing 10% FBS at 37°C in 5%
CO2, the monolayer SM3 cells were fixed and processed for
crystal violet staining. The results in Fig. 2, C and
D, also show a very good linear relationship between the
OD595 values of crystal violet nuclear staining and the
wide range of cell numbers (3.12 × 102-5 × 104 cells/well).
Monitoring proliferation rate of SM3 cells in culture.
We then established the seeding cell density for a reliable measurement
of the proliferation rate of SM3 cells. Nontransfected SM3 cells were
harvested from preconfluent cultures by digestion with 0.025% trypsin
in 0.02% EDTA solution and seeded into 96-well culture plates at 500, 1,000, and 1,500 cells/well in DMEM containing 10% FBS. Five identical
sets of cultures were started on five consecutive days and were stopped
altogether to obtain 30-, 54-, 78-, and 102-h cultures. The plates were
processed for crystal violet staining. Cell proliferation curves were
plotted to demonstrate the relationship to the initial seeding cell
density. The results in Fig. 3 show that
the SM3 cells cultured in 96-well plates from all of the three initial
densities had linear growth curves up to 102 h without changing
media. Accordingly, the proliferation rates of the transfected SM3
cells were examined under these conditions, except that the dispersion
of the transiently transfected SM3 cells was done by using Versene
solution to avoid enzymatic damage of the membrane proteins that may
affect the initial rate of cell proliferation.

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Fig. 3.
Growth curve of SM3 cells at various seeding densities. SM3 cells
were seeded in 96-well culture plates at 500, 1,000, and 1,500 cells/well. Cells were cultured in DMEM containing 10% FBS at 37°C
in 5% CO2 and measured for cell numbers by crystal violet
staining at a series of time points. Results plotted from means ± SD
of triplicate experiments show the duration of log-phase growth of SM3
cells under the cultural conditions used in the examination of
h2-calponin's effects on the rate of cell proliferation.
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Immunofluorescence microscopy.
Precleaned glass coverslips were coated with 0.1% gelatin and dried
under UV radiation before being placed in the culture dish. The
transfected SM3 cells were seeded to grow monolayers on the coverslips.
The coverslips with monolayer SM3 cells were collected at ~70%
confluence and washed with PBS. The cells were fixed with cold acetone
for 30 min. Immunofluorescence microscopy was carried out as described
previously (20) to examine the cellular localization of
the transfectively expressed h2-calponin. After blocking with 1% BSA
in PBS at room temperature in a humidity box for 30 min, the coverslips
were incubated with the rabbit anti-h2-calponin antibody RAH2 and a
mouse MAb against tropomyosin (CG3; provided by Dr. Jim J.-C. Lin,
University of Iowa; Ref. 25), alone or in combination, at
room temperature for 2 h. After washes with PBS containing 0.05%
Tween-20, the coverslips were stained with tetramethylrhodamine
isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG and/or
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG second
antibodies (both from Sigma) at room temperature for 1 h. After
final washes with PBS containing 0.05% Tween 20, the coverslips were
mounted on glass slides and examined under a Zeiss Axiovert 100H phase
contrast-epifluorescence microscope. A Plan-Neo phase fluorescence
×100 objective lens (oil; NA 1.30) was used for the photography of
both phase-contrast and fluorescence images. The TRITC and FITC
fluorescence images representing the localization of calponin and
tropomyosin, respectively, were selectively viewed through different
sets of filters (CZ915 and CZ909, respectively).
To determine the frequency of binuclear cells in the nontransfected and
transfected SM3 cell cultures, coverslips with preconfluent monolayer
cells were fixed and directly examined by phase-contrast microscopy as
described above.
Statistical analysis.
The quantitative data of cell proliferation are presented as means ± SD. Regression coefficients were calculated with Microsoft Excel.
Paired comparisons were carried out by Student's t-test to
examine the significance of difference.
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RESULTS |
Differentially regulated expression of h1- and h2-calponins during
postnatal development of mouse stomach and urinary bladder.
The expression of h1- and h2-calponins in the stomach and urinary
bladder smooth muscles of C57B6 mice during postnatal development was
examined by Western blot analysis. The results in Fig.
4 show that h1-calponin is expressed at
only low levels in the stomach and bladder muscles of neonatal mice but
upregulated during postnatal development to high levels in adult
stomach and bladder. In contrast to the postnatal upregulation of
h1-calponin, h2-calponin is expressed at high levels in the neonatal
mouse stomach and urinary bladder smooth muscles and downregulated
during postnatal development. Only a small amount of h2-calponin is
present in the adult tissues (Fig. 4). Furthermore, the levels of
either calponin isoform differ between the two smooth muscle organs.
Although the expression of h1- and h2-calponin appeared in a
complementary way, the quantitative relationship does not make up a
constant level of total calponin in the smooth muscle tissues. The
separate regulations of the h1 and h2 isoforms of calponin suggest that
they may play differentiated functions. These results are consistent
with previous studies showing that h1-calponin is expressed at a high
level in adult phasic smooth muscles (21, 32). On the
other hand, the high-level expression of h2-calponin in neonatal
stomach and bladder may indicate its role in tissue growth.

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Fig. 4.
Developmentally regulated expression of h1- and h2-calponins. Total
protein extracts from mouse stomach (A) and urinary bladder
(B) at a series of time points during postnatal development
were analyzed by SDS-PAGE and transferred to nitrocellulose membrane
for Western blotting with the anti-h1-calponin MAb CP3 and the
anti-h2-calponin MAb CP21. Purified mouse h1- and h2-calponins were
used as controls. Western blots show a developmental upregulation of
h1-calponin in both organs. Expression of h2-calponin is downregulated
during postnatal development with different patterns in the stomach and
urinary smooth muscles. An additional band with a molecular weight
slightly higher than that of h2-calponin was detected by the
anti-h2-calponin MAb CP21 in the 1-mo and 6-mo mouse bladder. Curves
from densitometry quantification of multiple Western blots (means ± SD; n = 3-4) summarize the changes in calponin
isoform expression.
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The CP21 MAb raised against cloned mouse h2-calponin showed no
cross-reaction to h1-calponin (Figs. 1 and 4). Interestingly, the
Western blot in Fig. 4B detected an additional protein band with a higher molecular weight than that of h2-calponin in both the
1-mo and 6-mo mouse urinary bladder. The amount of this protein seems
to be increased in the 6-mo vs. 1-mo bladder. Further studies are under
way to determine whether this protein is another calponin isoform or a
phosphorylation variant of h2-calponin, either of which would be
functionally significant in the development and activity of urinary
smooth muscle.
Regulated expression of h1- and h2-calponins in uterus smooth
muscle during pregnancy.
Western blots with the anti-h1-calponin MAb CP3 and the
anti-h2-calponin MAb CP21 showed high-level h1-calponin expression in
the nonpregnant and late-term uterus smooth muscle vs. high-level h2-calponin expression in the rapidly growing uterus of midterm pregnancy (Fig. 5). The high-level
expression of h1-calponin in prelabor uterus smooth muscle is
consistent with the potential role of h1-calponin in modulating the
contractility of smooth muscle. On the other hand, the high-level
expression of h2-calponin in rapidly growing uterus smooth muscle
suggests its role in regulating the actin cytoskeleton during smooth
muscle growth and cell proliferation.

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Fig. 5.
Regulated expression of h1- and h2-calponins in uterus
smooth muscle during pregnancy and involution. Total protein extracts
from the smooth muscle layer of mouse uterus were analyzed on SDS-PAGE
and transferred to nitrocellulose membrane for Western blotting with
the anti-h1-calponin MAb CP3 and the anti-h2-calponin MAb CP21.
A: blots show high-level expression of h1-calponin in the
uterus smooth muscle before labor vs. high-level expression of
h2-calponin in the rapidly growing midterm uterus. Purified h2- and
h1-calponins were used as controls. B: curves from
densitometry quantification of multiple Western blots (means ± SD; n = 3-4) summarize the changes in calponin
isoform expression.
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Transfective expression of h2-calponin inhibited the rate of cell
proliferation.
The Western blots in Fig. 6A
show that although h1- and/or h2-calponin are expressed in rabbit
vascular smooth muscle, the immortalized SM3 cells derived from rabbit
aorta have ceased the expression of calponin in preconfluent,
confluent, and differentiated cultures (37). This provides
a useful system to study the effects of calponin on cellular functions.
The role of h2-calponin in cell proliferation was investigated in SM3
cells through the transfective expression of h2-calponin. The Western
blots in Fig. 6B show that the h2 sense, but not antisense,
cDNA stable-transfected SM3 cells expressed a significant
amount of h2-calponin.

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Fig. 6.
Transfective expression of h2-calponin in SM3 cells.
A: total protein extract from rabbit blood vessels and SM3
cells before and after reaching confluence in culture were examined by
Western blot with anti-calponin antibodies (see Fig. 1). Results show
that although both h1- and h2-calponins are expressed in rabbit
vascular smooth muscle, the SM3 cell line derived from rabbit aorta has
ceased calponin expression. B: h2-calponin was expressed at
significant amounts in the h2-calponin sense, but not antisense, cDNA
stable-transfected SM3 cells. C: Western blot analysis on
total cellular proteins extracted from SM3 cells confirmed the
specificity of the anti-calponin RAH2 and the anti-tropomyosin (Tm) CG3
antibodies.
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The cell proliferation curves in Fig. 7
demonstrate a significantly decreased proliferation rate in the h2
sense vs. h2 antisense cDNA stable-transfected cells (P < 0.001). Initiated at the same number of cells, the number of h2
sense cDNA-transfected cells was only 33-42% of that of h2
antisense cDNA-transfected cells after 5 days of culture. The effect of
h2-calponin was independent of the presence or absence of G418 in the
culture medium.

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Fig. 7.
Inhibition of cell proliferation by stable transfective expression
of h2-calponin. SM3 cells stable-transfected with the sense or
antisense h2-calponin cDNA were seeded in 96-well culture plates in
DMEM containing 10% FBS and cultured at 37°C in 5% CO2
in the presence (A) or absence (B) of G418 (500 µg/ml). Cultures were stopped at a series of time points, and the
cell numbers were measured by crystal violet staining. Cell growth
curves were plotted from means ± SD of 4 experiments. Results
demonstrated a decreased proliferation rate in the h2 sense-transfected
vs. h2 antisense-transfected cells. *P < 0.001.
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Significant amounts of h2-calponin expression were also obtained in SM3
cells transiently transfected with the h2 sense, but not antisense,
expression vector (Fig. 8,
inset). The cell proliferation curves in Fig. 8 demonstrate a significantly decreased proliferation rate in the h2 sense vs. h2 antisense cDNA-transfected cells
(P < 0.001). The Western blot detected a transient
expression of h2-calponin in SM3 cells from 24 to 96 h after
transfection with the h2 sense cDNA. In contrast to the continuously
inhibited proliferation rate in the h2 sense stable-transfected SM3
cells (Fig. 7), the inhibition of cell proliferation was transient in
the transiently transfected SM3 cells depending on the expression of
h2-calponin (Fig. 8). After the cells ceased h2-calponin expression at
120 h after transfection, their proliferation rate returned
to a level similar to that of the h2 antisense cDNA
transfected and nontransfected SM3 cell controls (Fig. 8). In the
stable transfection experiments, the inhibitory effect of h2-calponin
was seen as early as 30 h after replating the cells (Fig. 7). This
more predominant effect seen in stable as opposed to transient
expression may be due to higher levels of h2-calponin as well as the
homogeneous expression exhibited in stable transfection in contrast to
the heterogeneous transient transfection. Nevertheless, these
results clearly demonstrate a direct relationship between h2-calponin
expression and decreased cell proliferation rate. Most importantly, the
results from transient transfection experiments represent a population
phenotype, excluding any potential effects from nonspecific changes in
individual stable-transfected cell lines.

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Fig. 8.
Inhibition of cell proliferation by transient transfective
expression of h2-calponin. SM3 cells were transiently transfected with
the h2 sense and h2 antisense expression vectors and cultured in DMEM
containing 10% FBS at 37°C in 5% CO2 for 18 h
before being replated in 96-well culture plates at 1,000 cells/well in
fresh medium. Cultures were stopped at a series of time points, and the
expression of h2-calponin was examined by Western blot with the
anti-h2-calponin antibody RAH2. Results in the inset show
transient expression of h2-calponin in the h2 sense, but not h2
antisense, cDNA-transfected SM3 cells. Cell numbers were measured at
these time points by crystal violet staining. Cell growth curves were
plotted from the means ± SD of 4 experiments. Dashed lines outline the
doubling time of the cultures. Growth curve of h2 antisense cDNA
transfected cells is similar to that of the nontransfected cells.
Although the initial proliferation rate of the h2-calponin-expressing
cells was not different from the controls when the growth was moderate,
the accelerating growth as that seen in the nontransfected and h2
antisense cDNA transfected cells was significantly delayed
(*P < 0.001). Cell proliferating rate of the h2
sense-transfected cells resumed ~24 h after h2-calponin ceased
expression.
|
|
It is worth noting that the proliferation rates of nontransfected and
h2 antisense cDNA-transfected SM3 cells began to accelerate 90 h
after replating (cell number doubling time shortened from 32-33 h
to 24 h). In h2 sense-transfected SM3 cells, the decrease in cell
proliferation rate seen 96 h after transfection (90 h after
replating and at least 72 h after the expression of h2-calponin became detectable) had prevented the acceleration of proliferation rate. It is possible that the expression of h2-calponin inhibited only
fast, but not slow or moderate, cell proliferation when transiently expressed in the SM cells. This selective effect may suggest that h2-calponin plays a role as a negative balancing factor to maintain a
physiological rate of cell proliferation. The inhibitory effect remained until 120 h after transfection, even though h2-calponin already had dramatically decreased expression by this time (Fig. 8).
This time lag in resuming proliferation rate may reflect the time
needed for the cells to recover from the effects of forced h2-calponin expression.
The initial seeding density of the cells did not affect the amount of
proliferation inhibition by h2-calponin. Experiments starting with 500, 1,000, or 1,500 cells/well yielded comparable results in both stably
and transiently transfected cultures (only 1,000 cells/well data are
shown in Figs. 7 and 8). In all experiments, the proliferation rates of
the h2 antisense cDNA-transfected cells and nontransfected cells were
almost identical (Fig. 8), indicating that the transfection procedure
and the integration of the vector DNA did not have a significant
nonspecific effect.
Association of h2-calponin to actin-tropomyosin filaments in
transfected SM3 cells.
Immunofluorescence microscopy with anti-h2-calponin antibody
demonstrates that the force-expressed h2-calponin localizes in the
stress fiber structures (Fig. 9). By
taking advantage of the fact that the rabbit anti-h2-calponin antiserum
and the anti-tropomyosin MAb are recognized by different second
antibodies with FITC or TRITC labels that can be distinguished by
viewing through different filter sets, double-staining
immunofluorescence microscopy clearly showed the colocalization of
h2-calponin and tropomyosin in the stress fibers (Fig. 9C).
Tropomyosin is a actin filament-associated protein (26),
and the results demonstrate the association of h2-calponin with the
actin filaments. The results also show a highly selective targeting of
the force-expressed h2-calponin to the actin stress fibers, because
very little background staining was observed. The association of
h2-calponin with the actin cytoskeleton suggests that its inhibitory
effects on the rate of cell proliferation may be based on an inhibition
of actin activity during cytokinesis. This hypothesis is supported by
the fact that no other protein in SM3 cell had significant reaction
with the anti-calponin RAH2 antibody and the anti-tropomyosin CG3
antibody used in the immunofluorescence localization (Fig.
6C).

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Fig. 9.
Association of h2-calponin with the actin-tropomyosin
filaments in transfected SM3 cells. SM3 cells stable-transfected with
h2 sense or h2 antisense cDNA were cultured on gelatin-coated
coverslips. Preconfluent monolayer cell samples were examined by
immunofluorescence microscopy with the rabbit anti-h2-calponin antibody
RAH2 and the mouse anti-tropomyosin MAb CG3, alone or as a mixture.
TRITC-conjugated anti-rabbit IgG and FITC-conjugated anti-mouse IgG
second antibodies, alone or as a mixture, were used to selectively
detect the localization of h2-calponin and tropomyosin, respectively.
Phase-contrast and fluorescence microscopic images were photographed.
A: TRITC and FITC fluorescence images can be selectively
obtained by using appropriate filter sets. B: stress fiber
association of both h2-calponin and tropomyosin was seen in the SM3
cells. C: double-antibody staining demonstrates a
colocalization of h2-calponin and tropomyosin.
|
|
Increased number of binuclear cells in SM3 cultures
force-expressing h2-calponin.
The number of binuclear cells was significantly increased in the h2
sense cDNA-transfected cells (25.08 ± 0.30%) vs. the h2 antisense cDNA-transfected (9.85 ± 0.44%) and nontransfected
(9.83 ± 0.30%) SM3 cultures (Fig. 10; P < 0.001). The increase in the number of
binuclear cells indicates that the forced expression of h2-calponin
does not directly reduce the rate of DNA replication to decrease cell
proliferation rate but rather inhibits the function of the actin
cytoskeleton during cytokinesis, which in turn results in slowed cell
division and proliferation. This hypothesis is consistent with the
results shown in Fig. 8, in which a time lag was present between the
expression of h2-calponin and the decrease of cell proliferation rate
as detected by the nucleus staining method.

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Fig. 10.
Increase of binuclear cells in h2-calponin expressing
cultures. Stable h2 sense and h2 antisense cDNA-transfected and
nontransfected SM3 cells were cultured on gelatin-coated coverslips.
Preconfluent monolayer cell samples were collected and fixed with
acetone for microscopic examination. Two thousand cells were examined
on each coverslip to calculate the rate of binuclear cells. Results
(means ± SD) summarized from 3 coverslips in each group demonstrated a
significantly increased number of binuclear cells in the h2
sense-transfected cultures (*P < 0.001).
|
|
Nuclear division is not commonly seen in mammalian cell division. The
increased frequency of cells with dividing nuclei in the cultures
force-expressing h2-calponin suggests that the suppression of actin
cytoskeleton function may prevent cytokinesis after
chromosome replication in the nucleus. Immunofluorescence
microscopy showed that in h2 sense cDNA-transfected binuclear SM3
cells, h2-calponin was enriched to surround the partially divided
nuclei, often forming a nuclear ring structure (Fig.
11). This actin filament-based nuclear ring structure is similar to the plasma membrane contractile ring, suggesting that the actin cytoskeleton may also play a role in nuclear
division. In contrast to the broad stress fiber distribution of
tropomyosin (Fig. 11A), the enriched association of
h2-calponin with the nuclear ring structure (Fig. 11B) may
imply its regulatory role in the nuclear division function of actin.

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Fig. 11.
Nuclear ring structure formed in the transfected SM3
cells by h2-calponin-containing stress fibers. Preconfluent monolayer
cultures of h2-sense cDNA-transfected SM3 cells on the coverslips were
fixed with acetone and examined by immunofluorescence microscopy with
the anti-tropomyosin CG3 MAb or the anti-h2-calponin antibody RAH2 and
TRITC-conjugated second antibody. Phase-contrast and immunofluorescence
(IFA) images of binuclear cells were examined and photographed. In
contrast to the extensive stress fiber association of tropomyosin
(A), h2-calponin was enriched around the nuclei
and participates in the formation of a nuclear ring structure
(B).
|
|
 |
DISCUSSION |
Independently regulated expression of h1- and h2-calponin.
The observation that the developmental expression of h1- and
h2-calponin in the bladder and stomach is regulated in opposite directions indicates their differentiated function. Upregulated expression of h1-calponin has been observed during smooth muscle differentiation and development (7, 11, 13, 32, 46), suggesting that it is involved in the functional maturation of myofilaments. A previous study observed increased expression of caldesmon in pregnant uterus smooth muscle, possibly playing a role in
suppressing contractility for the maintenance of pregnancy (49). Thus the decreased expression of h1-calponin in the
midterm pregnant uterus (Fig. 5) may also contribute to the suppression of uterus smooth muscle contractility. Western blots with the anti-h2-calponin MAb CP21 demonstrated that h2-calponin is expressed at
high levels in rapidly growing tissues such as the embryonic stomach
and bladder and downregulated during postnatal development (Fig. 4).
High levels of h2-calponin were also found in uterus smooth muscle
during early pregnancy (Fig. 5). The expression patterns of h2-calponin
may reflect its function in tissue growth and remodeling. The higher
levels of h2-calponin in rapidly growing and remodeling tissues support
its cytoskeletal function relating to cell proliferation. Therefore,
whereas h1-calponin may play a modulator role in tuning smooth muscle
contractility (29, 48), h2-calponin may play a regulatory
role in the function of the actin cytoskeleton in smooth muscle and
nonmuscle cells.
Potential role of h2-calponin in regulating the rate of cell
proliferation.
h1-Calponin's function as a regulatory protein for smooth muscle
contractility has been extensively investigated. However, the absence
of h1-calponin in rat aortic smooth muscle does not abolish
contractility (32). In fact, h1-calponin knockout mice remain normal in many physiological activities (29, 44).
Therefore, calponin is not an essential smooth muscle contractile
protein but rather a tuning element in smooth muscle
contractility. The specific function of h2-calponin, on the
other hand, is not yet known. Its presence in both smooth muscle and
nonmuscle cells indicates that it may have a cytoskeletal function.
Considering calponin's inhibitory activity on actin-myosin
interactions, h2-calponin may also play an inhibitory role in
regulating the functions of the actin cytoskeleton, such as
coordinating changes in cell shape and intracellular molecular
trafficking, both of which are critical events in cytokinesis
(15). Therefore, h2-calponin may act as a balancing
mechanism to maintain the physiological levels of actin filament
activity in both smooth muscle and nonmuscle cells. In the present
study we demonstrated that the expression of h2-calponin inhibits cell
proliferation, suggesting its regulatory role in cytokinetic
activities. The gene expression and activity regulation of h2-calponin
may contribute to normal organ development and the physiological growth
and remodeling of tissues. This hypothesis is supported by the
observations that significant amounts of calponin are associated with
the noncontractile actin cytoskeleton (34, 35) and that
forced expression of chicken gizzard (h1) calponin in cultured smooth
muscle cells and fibroblasts inhibits cell proliferation
(19). Also, h1-calponin knockout mice displayed enhanced
ectopic bone formation when they were stimulated by recombinant human
bone morphogenetic protein-2, once again suggesting calponin's function as a suppressor of cell proliferation. Calponin has been detected in the cytoplasm of human osteosarcoma cells, and the survival
rate of patients whose tumors exhibit calponin is significantly higher
than that of those whose tumors do not express calponin (50). Consistently, the h1-calponin knockout mice also had
an early onset of cartilage formation and ossification and
accelerated healing of bone fractures (51).
Interestingly, calponin is expressed notably less in leiomyosarcoma
cells than in normal smooth muscle cells (16).
Transfective expression of calponin in leiomyosarcoma cells
significantly reduced anchorage-independent growth and in vivo
tumorigenicity, indicating its function as a tumor suppressor (17).
h2-Calponin in the function of actin cytoskeleton.
Actin-myosin interaction-based cell motility is essential for
cytokinesis. The formation and function of a contractile ring during
the cell division is a clear example of this fact (3, 4, 36,
39). The contraction of the contractile ring is most likely
generated by the interaction between actin and myosin (2, 6, 22,
27). The actin cytoskeleton has been demonstrated to participate
in anchorage-dependent cell division (15), and actin-myosin interactions have been shown to power cell proliferation by driving cytoplasmic streaming. In vitro experiments have shown that
calponin inhibits the relative movements of actin and myosin (40). A calponin homologue in Xenopus has been
found to regulate cell motility during embryonic development by
inhibiting actin-myosin interactions (31). h2-Calponin's
association with the tropomyosin-actin filament also suggests that it
may inhibit the organization and motility of the actin cytoskeleton.
Thus calponin's role in regulating actin-myosin interaction and actin
cytoskeleton function may affect cytokinesis and the rate of cell proliferation.
During eukaryotic cell division, the nuclear membrane disintegrates to
allow for the mitotic separation of chromosomes. Although nuclear
division is often seen in cell cultures, the significantly increased
number of binuclear cells in h2-calponin-expressing cultures indicates
an inhibition of cytokinesis after chromosome replication. h2-Calponin
in the binuclear cells was concentrated around the nuclei, specifically
in a "nuclear ring" structure that, like the contractile ring, is
formed by actin filaments (Fig. 11). The association of h2-calponin to
the nuclear ring suggests that h2-calponin may inhibit the process of
nuclear division to prevent multiploidy in cells in which cytokinesis
was suppressed. Although the actin-tropomyosin stress fibers are
broadly distributed in the cell, the concentrated localization of
h2-calponin around the dividing nuclei indicates the presence of a
specialized domain of the actin cytoskeleton (Fig. 11) that is
regulated by h2-calponin. We have observed that calponin selectively
binds low-molecular-weight nonmuscle tropomyosin, suggesting a
potential functional correlation (unpublished results). Therefore, the
enrichment of h2-calponin in the nuclear ring may indicate that the
regulatory activity of h2-calponin may be targeted through the cellular
distribution of tropomyosin isoforms. Because calponin has been
observed to participate in the protein kinase C signaling pathway
(24), the function of h2-calponin in regulating the
activity of the actin cytoskeleton may play an important role in
maintaining physiological tissue growth and remodeling and deserves
further investigation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Jim J.-C. Lin for providing the CG3 MAb and Jill O. Jin for proofreading the manuscript.
 |
FOOTNOTES |
This work was supported by a grant from the March of Dimes Birth Defect
Foundation (J.-P. Jin).
Address for reprint requests and other correspondence:
J.-P. Jin, Dept. of Physiology and Biophysics, Case Western
Reserve Univ. School of Medicine, 10900 Euclid Ave., Cleveland, OH
44106-4970 (E-mail: jxj12{at}po.cwru.edu).
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.
First published September 4, 2002;10.1152/ajpcell.00233.2002
Received 21 May 2002; accepted in final form 3 September 2002.
 |
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Am J Physiol Cell Physiol 284(1):C156-C167
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