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INTRODUCTION |
Under many pathological conditions smooth muscle cells undergo
phenotypic modulation in which they change from a contractile quiescent
phenotype to a synthetic proliferative state. The phenotypic changes
observed depend on the severity of the injury and the specific smooth
muscle tissue involved (1-8). Smooth muscle cells in adult tissues are
normally quiescent, exhibit very low rates of division, and express
high levels of contractile proteins and myofilaments. These
differentiated, contractile smooth muscle cells are characterized by
the presence of unique isoforms of contractile proteins such as smooth
muscle
- and
-actin, myosin heavy chain, caldesmon, calponin,
SM22
, and telokin (4, 9-16). In vitro culture of smooth
muscle cells, in the presence of high levels of serum, results in a
marked decrease in the expression of many smooth muscle contractile
proteins, including smooth muscle myosin, smooth muscle actin,
h-caldesmon, calponin, smooth muscle myosin light chain kinase, and
telokin with a concomitant increase in the expression of non-muscle
contractile protein isoforms (2, 14,
17-19).1 A similar increase
in nonmuscle myosin has been reported in smooth muscle cells during
restenosis (20, 21). In contrast, in intimal cells from human coronary
arteries, it has been reported that there is reduced expression of SM2
smooth muscle myosin heavy chain without any detectable increase in
non-muscle myosin heavy chain B (5). These examples illustrate that
smooth muscle cells can exhibit a plethora of phenotypes ranging from
fully differentiated quiescent contractile cells to cells expressing
low levels of contractile proteins and proliferating rapidly (22).
In most in vivo models of vascular, airway, and intestinal
injury, smooth muscle cells are subject to mechanical and/or
cytological trauma resulting in exposure to growth factors and re-entry
into the cell cycle. Together these agents increase smooth muscle cell proliferation and cause phenotypic modulation altering the contractile properties of the muscle (22, 23). As both proliferation and phenotypic
modulation occur at the same time, it is difficult to assess the effect
of either process alone on the contractility of the muscle. To examine
carefully the relationships between smooth muscle cell proliferation,
differentiation, and contractility, we have generated a transgenic
mouse model in which visceral smooth muscle cells are induced to
proliferate in vivo following expression of an SV40 large
T-antigen oncogene. SV40 large T-antigen was specifically targeted to
smooth muscle cells using the smooth muscle-specific rabbit telokin
promoter. 2.4-Kilobase pair and 310-base pair fragments of the rabbit
telokin promoter have been shown to direct high levels of transgene
expression in smooth muscles of the gut, airways, reproductive and
urinary tract, and low levels of expression in vascular smooth muscle
(4, 24). In the current study we show that expression of T-antigen in
visceral smooth muscle cells induced cell proliferation without
significantly altering the contractile properties of the muscle. In
this transgenic model, smooth muscle cell proliferation was accompanied
by an increase in the non-muscle B myosin heavy chain isoform without any significant down-regulation of smooth muscle contractile protein isoforms. The increased smooth muscle cell proliferation did not significantly effect the contractile properties of the muscle. These
findings show that contractile smooth muscle cells can proliferate without down-regulating contractile protein expression. This suggests that the phenotypic modulation normally associated with proliferating smooth muscle cells following vascular or airway injury in
vivo or following in vitro culture of smooth muscle
cells occurs independently of proliferation.
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EXPERIMENTAL PROCEDURES |
Transgenic Mice--
The telokin promoter T2.4-SV40 large
T-antigen and T310-SV40 large T-antigen mice were described previously
(4, 24). Transgenic mice were generated in C3HeB/FeQ inbred embryos by the Indiana University Transgenic Mouse Facility using standard protocols.
Western and Northern Blotting--
Western and Northern blots
were performed as described previously (4, 25, 26).
Immunohistochemical Staining--
Tissues were collected from
transgenic mice, cryoprotected in 20% sucrose, phosphate-buffered
saline, overnight. Cryoprotected tissues were embedded in
tissue-freezing media (Triangle Biomedical Sciences, Durham, NC) and
frozen at
70 °. 8-µm cryosections were cut, fixed in 3.7%
formaldehyde in phosphate-buffered saline for 5 min, permeabilized in
0.2% Triton X-100 for 5 min, and then incubated with the appropriate
antibody for 2-6 h at 37 °C. Following extensive washing in 50 mM Tris, pH 7.6, 150 mM NaCl sections were
incubated with fluorescein conjugated to donkey anti-rabbit immunoglobulin G for 1 h.
Tritiated Thymidine Labeling--
Animals were given a
subcutaneous bolus injection of 200 µCi of
[3H]thymidine. 6 h after injection mice were
sacrificed, and tissues were rapidly dissected and treated for
immunohistochemical staining as described above. Following
immunostaining, sections were air-dried and dipped in photographic
emulsion (NTB2, Eastman Kodak Co.). Sections were exposed for 3-5 days
at 4 °C and developed according to the manufacturer's directions
(Kodak). Silver grains were visualized under bright field illumination.
Contractile Measurements--
Isolated rings (3-5 mm in length)
were cut from the proximal or distal colon of transgenic and wild type
mice. Rings were placed in a muscle bath and attached to an isometric
force transducer. Tissues were incubated at 37 °C, in
bicarbonate-buffered physiological saline solution bubbled with 5%
CO2, 95% O2. A resting tension of 1 g was
applied to each ring, and the rings were equilibrated for 1 h
prior to contractile measurements.
NADPH Diaphorase Staining--
Prior to NADPH diaphorase
staining, sections were fixed in 3.7% formaldehyde for 5 min,
permeabilized with 0.2% Triton X-100 for 5 min, and then washed in 10 mM sodium phosphate, pH 8.0, for 5 min. NADPH diaphorase
staining was then performed by incubating sections with 0.5 mM NADPH, 0.1 mM nitro blue tetrazolium, and 0.3% Triton X-100 in 10 mM sodium phosphate, pH 8.0, for
1 h at 37 °C. Following staining, sections were washed in 10 mM sodium phosphate, air-dried, and mounted in Permount (Sigma).
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RESULTS |
Targeted Expression of SV40 Large T-antigen to Visceral Smooth
Muscle Results in Megacolon--
The most prevalent phenotype observed
in mice expressing SV40 large T-antigen in visceral smooth muscle was a
gross enlargement of the colon either alone or in conjunction with
enlargement of the entire intestinal tract (Table
I). The enlarged colon was impacted with
digesta and in all cases resulted in premature death. Most animals died
soon after weening with the exception of one female (T310-T-antigen)
transgenic animal from which a line was established. The onset of the
intestinal pathology was delayed in the animals from this founder,
potentially due to the effect of the transgene integration site on the
timing of transgene expression. The transgene integrated into the X
chromosome in this line and telokin-T-antigen transgene expression is
not detectable until the animals are approximately 4 weeks old. Mice
from this transgenic line all develop megacolon after 4-6 months; the
phenotype is 100% penetrant for both male and females and persistent
through greater than 10 generations. Although the extent of the
enlarged portion of the colon is variable from animal to animal, within the line, the greatest enlargement is always seen in the proximal colon
closest to the cecum (Fig. 1).
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Table I
Phenotypes of telokin-T-antigen transgenic mice
The phenotypes of founder animals in which smooth muscle-specific
expression of SV40 large T-antigen driven by either 2.4-kilobase pair
(Telokin 2.4 T-Antigen) or 310-base pair (Telokin 310 T-antigen)
fragments of the telokin promoter are shown.
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Fig. 1.
Telokin-T-antigen transgenic mice develop
megacolon. Shown are the dissected digestive tracts (from stomach
to rectum) from a telokin-T-antigen transgenic animal
(TRANSGENE) and a wild type litter mate (WILD
TYPE). P indicates the proximal colon. D
indicates the distal colon.
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Analysis of hematoxylin- and eosin-stained cross-sections revealed that
the enlarged proximal colon of transgenic animals had a much larger
diameter than the proximal colon of wild type mice (Fig.
2A). Analysis of
cross-sections of the colon revealed that the thickness of the enlarged
proximal colon wall was grossly similar in transgenic and wild type
animals (Fig. 2B.). In contrast, the density of smooth
muscle cells in the transgenic colon was significantly higher in
transgenic animals as compared with wild type animals.

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Fig. 2.
Megacolon results in an increase in the
circumference of the colon. Hematoxylin- and eosin-stained
cross-sections through the proximal colon of wild type and
telokin-T-antigen transgenic animals are shown. A, low
magnification. B, high magnification showing the position of
the longitudinal muscle (LM), circular muscle
(CM), submucosa (SM), muscularis mucosa
(MM), and epithelial cells of the mucosa
(M).
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T-antigen-induced Megacolon Is Distinct from Other Mouse Genetic
Models of Megacolon--
In human megacolon resulting from
Hirschsprung's disease and in several mouse models of megacolon, the
lack of intramural ganglia in the distal colon results in constriction
of this region causing megacolon (27). To determine if a similar
disruption of innervation could account for the phenotype of the
telokin-T-antigen transgenic mice, enteric ganglia were visualized by
NADPH diaphorase staining of tissue sections (Fig.
3). This analysis demonstrated that
unlike other mouse models of megacolon the telokin-T-antigen transgenic
animals exhibited enteric ganglia in both the proximal and distal
colon. No agangliosis was evident in these animals. Similar results
were seen when ganglia were visualized by neurofilament staining (data
not shown).

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Fig. 3.
Colon of telokin-T-antigen transgenic mice
contain enteric ganglia. Cross-sections of distal and proximal
colon from wild type (WT) and telokin-T-antigen transgenic
(TG) animals stained for NADPH diaphorase activity. Positive
nerves are stained purple. Several myoenteric ganglia are
indicated by the arrows. Scale bar indicates 50 µm.
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Expression of SV40 Large T-antigen in Visceral Smooth Muscle
Induces Smooth Muscle Proliferation--
Although the wall of the
proximal colon was not overtly thickened, the circular and longitudinal
muscle layers displayed hypercellularity (Fig. 2B). There
was a 4-5-fold greater nuclear density in the muscle layers of
transgenic animals as compared with wild type animal, suggesting that
the smooth muscle cells within these layers were proliferating. To
determine directly if expression of T-antigen in visceral smooth muscle
cells induces cell proliferation, animals were labeled with
[3H]thymidine for 6 h prior to sacrifice. Analysis
of sections obtained from these animals demonstrated that the
telokin-T-antigen transgene induced [3H]thymidine
incorporation into the nuclei of smooth muscle cells (identified by
smooth muscle myosin staining) of the circular, and longitudinal muscle
layers throughout the intestinal tract, indicating that these cells
were proliferating (Fig. 4). In contrast, wild type animals exhibited very low levels of smooth muscle cell proliferation as evidenced by no [3H]thymidine-positive
smooth muscle cells in many sections. Most of the
[3H]thymidine-positive nuclei were confined to the
epithelial cells in the crypts of the colon of wild type animals (Fig.
4).


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Fig. 4.
Visceral smooth muscle targeted expression of
SV40 large T-antigen induces smooth muscle cell proliferation.
Animals were labeled with tritiated thymidine for 6 h prior to
sacrifice. The digestive tract was dissected from the animals, and
segments were processed for immunofluorescent analysis as described
under "Experimental Procedures." Cross-sections of gut were stained
with antibodies specific for either smooth muscle myosin
(SM2) or T-antigen (TAG), both of which are only
expressed in smooth muscle cells. Some sections were also stained with
antibodies to non-muscle myosin heavy chain B (NMHCB; a
generous gift form Dr. Robert Wysolmerski) as indicated. Stained
sections were then dipped in photographic emulsion to identify nuclei
that had incorporated tritiated thymidine. Staining was visualized
under epifluorescence with additional back light illumination to
visualize both the antibody staining (green) and the
tritiated thymidine-positive nuclei (black granules).
Sections shown are from distal colon (DISTAL), proximal
colon (PROX), ileum, jejunum (JEJ), and stomach,
as indicated. Scale bars represent 50 µm. Representative
sections are shown from a 6-month-old telokin-T-antigen transgenic
mouse that displayed overt megacolon, from a 6-month-old wild type
mouse, and from a 2-month-old telokin-T-antigen transgenic animal that
displayed no overt pathology, as indicated.
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Increased Smooth Muscle Cell Proliferation Occurs Prior to the
Development of Megacolon--
To determine if the increase in smooth
muscle cell proliferation observed in the animals with megacolon was a
secondary consequence of the pathology, proliferation was examined in
2-month-old transgenic animals prior to the development of the
intestinal pathology. Proliferation was determined by tritiated
thymidine uptake into the nuclei of smooth muscle cells. This analysis
revealed that visceral smooth muscle cells throughout the
gastrointestinal tract exhibited high rates of proliferation prior to
the development of megacolon (Fig. 4). Despite increased rates of
smooth muscle cell proliferation in all regions of the gut, no major
histological changes were consistently observed in any region except
for the colon. The amount of smooth muscle cell proliferation in the
circular muscle layer of proximal and distal colon of wild type and
2-month-old telokin-T-antigen transgenic animals was quantitated by
determining the ratio of 3H-positive nuclei to total nuclei
in this layer. The muscle layers were identified by counterstaining for
smooth muscle myosin. This analysis demonstrated that in transgenic
animals there was a large increase (10-60-fold) in proliferation in
the smooth muscle cells in both the proximal and distal colon as
compared with wild type animals (Table
II). Calculation of the proliferative
index obtained from analysis of several sections from four 2-month-old
transgenic animals also revealed that there was no significant
difference in the rate of proliferation in the proximal as compared
with distal colon (6.35 ± 3.3% in proximal as compared with
6.5 ± 1.9% in distal; Table II).
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Table II
Proliferative index of colonic smooth muscle cells
Tissues were obtained from animals that were labeled with tritiated
thymidine 6 h prior to sacrifice. Cryosections cut from the proximal
and distal colon were stained with antibodies specific for smooth
muscle myosin, counter-stained with Hoechst, and then processed for
emulsion autoradiography to identify 3H-positive nuclei.
Hoechst staining was used to count the total number of nuclei in the
smooth muscle layer in each microscope field. The smooth muscle myosin
staining aided in the identification of the smooth muscle layers.
3H-positive nuclei were visualized by bright field
illumination. Data were obtained from several microscope fields
obtained from at least three sections cut from each tissue. Data
obtained from four transgenic (TG) and four wild type (WT) animals are
shown.
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Identification of the Physiological Defect Resulting in
Megacolon--
In all known models of megacolon the pathological
enlargement of the colon occurs subsequent to blockage of the distal
colon and reflects the stretch imposed on the wall of the colon by the continued movement of digesta into the obstructed region. There is no
obvious physical obstruction in the colon of telokin-T-antigen transgenic animals, suggesting that, similar to other animal models of
megacolon, it is likely that altered contractility results in the
obstruction. However, in contrast to other mouse models of megacolon,
the impaired contractility is not a result of agangliosis of the distal
colon (Fig. 3). To investigate possible mechanisms that cause the
megacolon in the telokin-T-antigen transgenic animals, transgenic mice
were examined at 2 months of age prior to the development of any gross
intestinal pathology. Two possible models can be envisioned by which
the contractility of the colon could be altered such that digesta
accumulates in the proximal colon. Either the contractility of the
distal colon is increased resulting in a constriction that prevents the
movement of digesta out of the proximal colon or the contractility of
the proximal colon could be decreased such that it is unable to move
digesta to the distal colon. Several physiological parameters were
examined in order to identify the cause of the motility defect that
results in the obstruction of the colon.
Maximal Contractile Responses of Colonic Muscle Rings Are Not
Significantly Different between Transgenic and Wild Type Mice--
To
determine the effect of increased smooth muscle cell proliferation on
the physiological function of the muscle, colonic muscle rings were
isolated and their contractile properties examined. Three rings of
proximal and 3 rings of distal colon were analyzed from each of 6 animals. Maximal contractions in response to KCl (80 mM),
electrical (20 Hz, 80 V), and carbachol (10
4
M) stimulation, normalized to the cross-sectional area of
the circular muscle layer in each sample, were not significantly
different between wild type and transgenic animals (Fig.
5, A-C). In contrast, both
wild type and transgenic distal colon exhibited higher maximal contractions to electric and carbachol stimulation as compared with
proximal colon (p < 0.05; Fig. 5, B and
C).

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Fig. 5.
The contractility of colonic muscle from
telokin-T-antigen transgenic mice is similar to wild type. Rings
of colon (approximately 4 mm in length) were cut from the proximal and
distal colon of 2-month-old telokin-T-antigen transgenic
(TG, hatched bars) and wild type (WT,
solid bars), age-matched animals. Muscles were hung in a
myobath, and their maximal contractile responses to various agonists
was measured as described under "Experimental Procedures."
A, contractions were initiated by addition of 80 mM KCl. B, contractions were initiated by
electrical field stimulation (80 V, 20 Hz, 10 s). C,
contractions were initiated by addition of 10 4
M carbachol. Contractions, measured in grams, were
normalized to the cross-sectional area of the circular muscle layer in
each ring (as this is the muscle layer contributing to the
contraction). Three distal and three proximal rings were analyzed from
each animal. The data presented represents the mean ± S.E.
obtained from 6 animals. Data were statistically analyzed by analysis
of variance to identify groups that were statistically different from
each other. Statistically significant differences (p < 0.05) are indicated on each graph, together with their p
values. Data obtained from wild type animals are indicated by
solid bars, and data obtained from transgenic animals are
shown by hatched bars. D, the thickness of the
circular muscle layer of proximal and distal colon was determined by
morphometric analysis of hematoxylin- and eosin-stained sections using
Metamorph software. Data represent the mean ± S.E. obtained from
a total of at least 15 sections obtained from 4 to 6 different animals.
Statistical differences were analyzed by analysis of variance; values
having a p value >0.05 were considered not significant.
Data obtained from wild type animals is shown by solid bars
and from transgenic animals by hatched bars. E
and F, dose-response of muscle rings to carbachol. Three
distal and three proximal rings were analyzed from each animal. The
data presented represent the mean ± S.E. data obtained from 3 animals. Data are expressed as a percentage of the maximal
carbachol-induced contraction. E, samples obtained from
proximal colon. F, samples obtained from distal colon.
Closed symbols indicate data from wild type animals and
open symbols from transgenic animals, as indicated.
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The contractile responses to carbachol were further evaluated to
determine if the sensitivity to agonist stimulation was altered in the
transgenic animals. In both proximal and distal colon the carbachol
dose-response curves of transgenic animals were shifted to the right as
compared with wild type animals, indicating that muscle from transgenic
animals was less sensitive to agonists compared with muscle from wild
type animals (Fig. 5, E and F). The concentration
of carbachol required to produce a half-maximal contraction increased
from 4.4 × 10
7 to 9 × 10
7
M in distal colon and from 8 × 10
7 to
1.7 × 10
6 M in proximal colon.
The Circular Muscle Layer in the Distal Colon of Telokin-T-antigen
Transgenic Animals Is Significantly Thicker Than in Wild Type
Animals--
The thickness of the circular muscle layer of the
proximal and distal colon of 2-month-old transgenic and wild type mice
was determined by morphological analysis of hematoxylin- and
eosin-stained sections of the colon, using Metamorph Image analysis
software (Fig. 5D). Results from this analysis demonstrated
that in both wild type and transgenic animals the circular muscle layer
in the proximal colon is significantly thicker than in the distal colon
(p < 0.01, Fig. 5D). In addition, the
circular muscle layer of the distal colon of transgenic animals was
significantly thicker than the wild type animals (p < 0.01). In contrast, there was no significant difference in the proximal
colon between transgenic and wild type animals.
The Distal Colon from Telokin-T-antigen Transgenic Animals Is More
Contractile per Unit Length Than Distal Colon from Wild Type
Animals--
The data shown in Fig. 5 demonstrate that the maximal
contractile responses of transgenic and wild type colon are similar when normalized to the cross-sectional area of circular muscle. However, the circular muscle in the distal colon of transgenic mice was
significantly thicker than in wild type animals (Fig. 5D).
Together these data suggest that per unit length the transgenic distal
colon is more contractile than wild type. When the maximal forces
obtained from colonic rings are expressed per unit length, it is
apparent that although there is little change in the contractility of
wild type and transgenic proximal colon, the transgenic distal colon is
significantly greater than wild type distal colon. In addition, the
contractility of wild type proximal colon is greater than the
contractility of wild type distal colon (Table
III). This pattern is generally reversed
in telokin-T-antigen transgenic animals in which the distal colon is
more contractile in response to electrical and KCl stimulation than the
proximal colon.
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Table III
Maximal contractility of colonic rings normalized per unit length
The maximal contractile responses to KCl, electrical, and 10 4
M carbachol stimulation, normalized to cross-sectional
area, are shown in Fig. 5, A-C. To determine the amount of
force per unit length of colon, the mean values shown in Fig. 5,
A-C were multiplied by the mean thickness of the circular
muscle layer, shown in Fig.
5D.
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T-antigen-induced Smooth Muscle Proliferation Does Not Effect
Smooth Muscle Contractile Protein Expression--
Phenotypic
modulation of smooth muscle, resulting in alteration of the expression
of contractile protein isoforms, is a common feature associated with
smooth muscle cell proliferation. To examine the expression of smooth
muscle contractile proteins in telokin-T-antigen transgenic mice,
Western blot analysis was performed on extracts obtained from 4 transgenic and 4 wild type mice (Fig. 6,
A and B). Autoradiographs were scanned, and the
density of each band was quantitated. This analysis revealed that
expression of the non-muscle B myosin heavy chain isoform in proximal
and distal colon of telokin-T-antigen transgenic animals was increased
approximately 10-fold (proximal, p < 0.05; distal,
p < 0.001) as compared with expression in colon from
wild type animals (Fig. 6, A and B). In contrast,
a small (15%) although statistically significant decrease (proximal,
p < 0.01; distal, p = 0.04) of the SM2
smooth muscle myosin heavy chain isoform was observed in colon from
transgenic as compared with wild type animals. The expression level of
other contractile proteins was not significantly different between wild type and transgenic animals. Comparison of telokin and T-antigen protein expression between proximal and distal colon of transgenic animals revealed no statistical differences (data not shown).

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Fig. 6.
Smooth muscle contractile proteins are not
down-regulated in telokin-T-antigen transgenic smooth muscle.
Tissue extracts were prepared from samples of proximal (A)
and distal (B) colon from 2-month-old wild type and
telokin-T-antigen transgenic mice (n = 4). Transgenic
mice did not display any overt megacolon. 15 µg of each extract were
analyzed by Western blotting with specific antibodies to several
contractile protein isoforms. Antibodies used were specific for smooth
muscle myosin heavy chain SM1 (SM1), smooth muscle myosin
heavy chain SM2 (SM2), non-muscle myosin heavy chain A
(nmHCA), non-muscle myosin heavy chain B (nmHCB),
h- and l-caldesmon (hCALD and l-CALD), 130-kDa
smooth muscle myosin light chain kinase (smMLCK), and
220-kDa non-muscle myosin light chain kinase (nmMLCK),
telokin, SM22 , and smooth muscle -actin. Proteins shown in
brackets were not expressed at detected levels in the
extracts analyzed. The positions of molecular mass markers are
indicated at the left of the blots.
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DISCUSSION |
Specific, targeted expression of SV40 large T-antigen to visceral
smooth muscle cells stimulates the cells to re-enter the cell cycle and
results in proliferation of contractile smooth muscle cells, without
inducing de-differentiation. This leads to an increase in the
contractility of the distal colon and results in the development of
megacolon. It is likely that the hyper-contractile distal colon creates
a restriction to the passage of digesta through the colon, causing the
proximal colon, oral to the restriction, to enlarge producing the
megacolon phenotype.
Visceral smooth muscle hypertrophy is a common pathology of many human
diseases (23). Diseases that result in partial obstruction of the gut,
such as Hirschsprung's disease, cause a marked smooth muscle cell
hypertrophy in cells oral to the obstruction, leading to megacolon.
Hypertrophy of the gut following partial obstruction is accompanied by
a large increase in the circumference of the intestine oral to the
obstruction; this results from increased smooth muscle cell
proliferation, in addition to cellular hypertrophy (23). Based on
ultrastructural investigations it was shown that the proliferating
cells maintained a full complement of myofilaments and specialized
structures suggesting that they remained fully differentiated.
Similarly, hypertrophy of smooth muscle cells in the urinary bladder
occurs following partial obstruction of the outflow tract, and
hypertrophy of smooth muscle cells in the trachea and bronchi are seen
in chronic asthmatic patients. The hypertrophy of the gut in
telokin-T-antigen transgenic mice, although superficially similar to
the hypertrophy resulting from experimental constriction of the gut, is
morphologically distinct in that most of the increase in muscle volume
results from hyperplasia rather than hypertrophy of smooth muscle cells
(Figs. 1 and 2). Although the size of the smooth muscle cells has not
been directly determined in the T-antigen transgenic mice, there is a
dramatic 4-5-fold increase in the nuclear density of the circular
muscle layer, with a relatively small increase in its thickness (less
than 2-fold). These physical constraints imply that the smooth muscle
cells in the telokin-T-antigen transgenic animals are likely to be
smaller than cells from wild type animals. Although the contribution of smooth muscle cell hyperplasia to the phenotype of the muscle in
experimentally induced gut hypertrophy is not well understood, several
investigators have shown that proliferating smooth muscle cells can be
found in the hypertrophic gut (reviewed in Ref. 23). Similar to other
experimental and genetic models of gut hypertrophy, the
telokin-T-antigen transgenic mice model also results in a large
increase in muscle volume without altering the stratified architecture
of the gut. This demonstrates the remarkable ability of the intestine
to maintain the normal organization of the external muscle, mucosal, or
submucosal layers even in the presence of a dramatic increase in smooth
muscle cell proliferation directed by a viral oncogene.
In previous animal models of visceral smooth muscle hypertrophy, a
partial obstruction of the colon placed a mechanical strain on the wall
of the gut triggering the hypertrophic response. In the
telokin-T-antigen transgenic animals, mechanical strain also results
from a constriction of the colon. The subsequent remodeling and
enlargement of the gut is likely to occur as a result of this mechanical strain. Unique to the telokin-T-antigen transgenic animals
an increase in smooth muscle cell proliferation is likely to cause the
initial constriction. Morphological measurements of the distal colon in
transgenic animals show that the circular muscle layer is significantly
thicker than in wild type animals. This results in the increased
contractility of the distal colon, and it is likely that it is this
increased contractility that presents a constriction to the flow of
digesta throughout the colon. The increased pressure of the luminal
contents on the oral side of the constriction will then provide the
mechanical strain needed to induce remodeling and hypertrophy of the
gut. Thus, in addition to loss of relaxing innervation of the colon, as
seen in aganglionic megacolon, smooth muscle cell hyperplasia leading to muscle hyper-contractility can also lead to megacolon. These results
suggest that an increase in the rate of smooth muscle cell
proliferation in the wall of the gut may play a role in the etiology of
diseases, such as idiopathic megacolon, that result in megacolon
without loss of neuronal innervation (28).
The telokin T-antigen transgenic model provides a novel system in which
to study the effects of smooth muscle cell proliferation on the
contractile properties of muscle in vivo. In young
transgenic animals prior to the intestinal remodeling, smooth muscle
cells in the wall of the intestine are proliferating rapidly with a proliferative index of 6% as compared with <0.8% in wild type animals (Table II). Analysis of intestinal contractility in these animals provides a novel system to investigate the effects of increased
smooth muscle cell proliferation alone, in the absence of overt
mechanical stimuli, on contractility. Results from this analysis
clearly show that, although the colonic smooth muscle cells from
telokin-T-antigen transgenic animals are proliferating, this does not
effect the maximal contractile response of the muscle to agonists.
Neither receptor-independent (KCl) nor receptor-dependent (carbachol, electric stimulation) responses were significantly different between wild type and transgenic animals (Fig. 5). The only
difference observed was a small rightward shift in the dose-response curve to carbachol in the transgenic animals (Fig. 5, E and
F). Together with these physiological findings, biochemical
analysis of contractile protein expression revealed that there was no
significant down-regulation of contractile protein expression in the
proliferating colon (Fig. 6). These findings are consistent with
previous studies on the lethal spotted mouse model of megacolon, in
which no decrease in contractile protein expression was observed in the
hypertrophic colon (29). The most significant change in contractile
protein expression observed in telokin-T-antigen transgenic animals was an increase in the non-muscle B myosin heavy chain isoform. In contrast, no increase in the expression of the non-muscle or embryonic isoforms of l-caldesmon, myosin light chain kinase, or nonmuscle myosin
heavy chain A was observed. This suggests that the proliferating muscle
has not simply reverted to an earlier embryonic state. A similar
increase in non-muscle myosin B has been observed in many different
models of smooth muscle cell proliferation (6, 30, 31), supporting the
hypothesis that this embryonic myosin isoform may be necessary for
smooth muscle cell proliferation (32).
Results presented provide direct evidence that visceral smooth muscle
cells can proliferate in vivo without down-regulating contractile protein expression or without affecting the contractile properties of the muscle. These results imply that the phenotypic modulation of smooth muscle cells that occurs following vascular or
airway injury or when cells are cultured in vitro is likely to be regulated independently of changes in cell proliferation. In
support of this concept, it has been shown that protein kinase G
promotes differentiation of cultured vascular smooth muscle cells
without altering their ability to proliferate (33). These observations
support a growing body of evidence suggesting that the proliferative
potential of a vascular or visceral smooth muscle cell is not
obligatorily linked to its' state of differentiation.