Heterogeneous lsoactin Gene Expression in the Adult Rat Gastrointestinal Tract REBECCA Department
A. LIDDELL,
MARK
SYMS,
and
KIRK M. MCHUGH
of Anatomy, Thomas Jefferson University, Philadelphia, Pennsylvania
BacMround: Normal gastrointestinal development is a complex process involving the precise integration of multiple cell types. To gain a better understanding of these processes, the present study examined isoactin gene expression in the adult rat gastrointestinal tract. Methods: Northern blot analysis was performed on specified segments of the adult rat esophagus, stomach, small intestine, cecum, colon, rectum, and anus using actin isoform-specific complementary DNAs for all six vertebrate isoactins. Results: Smooth muscle and cytoplasmic isoactins were heterogeneously coexpressed in a segment-specific manner throughout the gastrointestinal tract. In addition, striated muscle isoactin expression was also detected in segments of the adult rat esophagus, stomach, colon, cecum, rectum, and anus. Histological analysis indicated that the adult rat esophagus, stomach, and anus contained significant quantities of skeletal muscle, providing a source for the striated muscle isoactins detected in these gut segments. A similar source of striated muscle isoactin expression in the cecum, colon, and rectum was not identified. Both coordinate and independent regulation of isoactin gene expression was observed in the gastrointestinal tract, although distinct patterns of autoregulation were absent. Conc/usions: This study represents the first complete analysis of isoactin gene expression in the adult rat gastrointestinal tract and provides the basis for future studies designed to investigate the factors responsible for these processes.
from
six independent
tinct
temporal
ment
and in the adult.
pression
N
make
embryonic germ layers as well as a precise series of spatial reorderings including embryonic flexion and
rotation of the midgut. Recent studies in our laboratory have begun to analyze the molecular mechanisms responsible for some of these complex processes by examining isoactin gene expression in the developing and mature gastrointestinal tract of the rat and mouse. Actin is expressed as six distinct isoforms in higher vertebrates that include the P-cytoplasmic, y-cytoplasmic, ‘y-smooth muscle, a-smooth muscle, u-skeletal, and a-cardiac. These six actin isoforms are derived
the
spatial
patterns
These
various
actin
for the differentiation of tissue and cell types.
Prior
studieslM3 have
isoactins
are ubiquitously
throughout
shown
of the muscle
tive
to be limited
tends
phenotypes.
The smooth
ily coexpressed testinal
system.
They
muscle
isoactin
the
during
that
the cytoplasmic
of a
in all cell types
isoactins
In con-
is more restric-
to specific
muscle
isoactins
muscle
However,
include
maturation
cell
are primar-
cells of the gastroin-
tract, vascular
do exist for the expression tins.
of ex-
excellent
and
muscle
in smooth
tract, urogenital
spiratory
isoforms
and into adulthood.
trast, expression and
develop-
patterns
coexpressed
development
in dis-
during
distinct
variety
several
system,
and re-
additional
of the smooth expression
caveats
muscle
isoac-
of the cl-smooth
the early stages of striated
mus-
cle myogenesis
as well as in a variety
of myofibroblas-
tic cell type?
and the expression
of the y-smooth
muscle
isoactin
Coexpression shown heart
in developing
of the striated
to be limited and skeletal
Studies
muscle
family showed
the stomach
isoactins
has been
and
expression
in the rat and mouse. that the cytoplasmic and mature
these studies
that the striated
sperm.” mature
have characterized
were the primary
in the developing
mature
muscle.‘0-‘2
and tissue-specific
multigene
addition,
and
to the developing
in our laboratory’
velopmental
cle isoactins gastrointestinal development requires the of multiple cell types from all three interaction
and
markers
analysis ormal
genes that are expressed
the de-
of the actin Northern
blot
and smooth
mus-
actin isoforms gastrointestinal
expressed tract. In
also made the novel observation
muscle
and intestine
isoactins
were coexpressed
of the developing
in
and ma-
ture rat. In situ analysis (K. M. McHugh, manuscript in preparation) indicated that expression of the smooth muscle isoactins was limited to the smooth muscle cells of the gastrointestinal tract, whereas additional studies’-3 suggested that the cytoplasmic isoacAbbreviations used in this paper: tcRNA, total cellular RNA. 0 1993 by the American Gastroenteroiogical Association 0016-5085/93/$3.00
348
LIDDELL
ET AL.
GASTROENTEROLOGY
tins were coexpressed
in all cell types found
developing
and mature
cise nature
and location
the striated
muscle
tract remains These
and
isoactin
studies
and
study
striated
colon,
cellular
RNA
segments,
rectum,
(1) to further of the actin
rat gastrointestinal and
muscle
of adult rat esophagus,
cecum,
The pur-
expression
rat gastrointestinal
tract
location
isoactin tract.
of the
expression
Specified
stomach,
seg-
small intestine,
and anus were harvested.
was isolated
Northern
from
blotted,
complementary
results
showed
distinct
specific
patterns
with
(cDNA)
organ-specific
of isoactin
Total
each of these bowel
and probed
isoactin-specific
all six
probes. and
through-
out the adult rat gastrointestinal
tract. In addition,
precise
source
expression
muscle
isoactins
was further information adult
in the adult
the
of striated
rat gastrointestinal
tract
characterized. This study provides regarding isoactin gene expression
novel in the
rat gastrointestinal
complexity
The
segment-
gene expression
of the “ectopic”
of
gene expres-
adulthood.
the extent
reported
in the adult
patterns development.
was twofold:
in the adult
(2) to determine
ments
into
the heterogeneous family
previously
of
that the rat
of isoactin
also maintained
characterize
shown distinct
throughout
patterns
pose of the present multigene
displayed
expression
organ-specific
sion were
expression
in the rat gastrointestinal
had clearly
intestine
gene
These
the
tract. The pre-
of the “ectopic”
isoactins
No. 2
to be clarified.
earlier
stomach
gastrointestinal
within
Vol. 105,
tract
previously
and suggests
a level of
unreported.
Materials and Methods Animals Both
male
(Zivic
Miller,
Adult
rats were
intestine,
cecum,
esophagus, cally ments
and
and the esophagus, rectum,
further in Figure
for RNA
rats tissues.
stomach,
and anus were excised.
small intestine,
and
as outlined
Sprague-Dawley
colon,
stomach,
measured
adult
PA) were used to obtain
killed,
segments were pooled ately frozen in liquid needed
female
Zielienople,
small The
and colon were specifi-
subdivided
into
1. Specific
organs
distinct
seg-
and organ
from approximately 14 rats, immedinitrogen, and stored at -70°C until
isolation.
cDNA
probes
specific
characterized
1.
Schematic
segments
was specifically measured ment
and published.
have
correspond
2 (~2),
stomach
for all six actin isoforms
representation
jejunum
of the specific gastrointestinal
isolated from the adult rat. Each gastrointestinal
segments
DNA Probe Construction been previously
Figure
to esophagus
esophagus
segment (J),
and excised. segment
2 (s2),
stomach
ileum (I), cecum
COlOn (TC), descending
(cE),
segment
The adult rat gastrointestinal
segment 3 (~3),
1 (EI),
stomach
segment ascending
3 (s3),
esophagus segment
seg1 (sI),
duodenum
(D),
colon (AC), transverse
COlOn (DC), rectum (R), and anus (A).
These include
a 125-base pair (bp) rat y-smooth muscle isoactin-specific,” a 187-bp rat a-smooth muscle isoactin-specific,” a 404-bp human P-cytoplasmic isoactin-specific,‘3 a 365-bp
tin-specific14 probe. All six of these probes are derived from the 3’ untranslated regions of their respective isoactin cDNAs and are of similar length and G/C content. Isoac-
human y-cytoplasmic isoactin-specific,‘3 a 136-bp human a-skeletal isoactin-specific,‘4 and a 171-bp &cardiac isoac-
tin-specific mid DNA
cDNA insert fragments were isolated from plasusing the appropriate restriction enzymes. Insert
August
ISOACTIN
1993
by standard agarose gel electrophoresis and electroelution using an Elutrap apparatus (Schleicher & Schuell, Keene, NH). A total of 50 ng of each insert was labeled by random priming to a specific activity of at least 1 X lo8 cpm * pg-’ * mL-’ before use in Northern blot analysis. DNA
was purified
RNA Isolation and Northern Blot Analysis
were stripped
mmol/‘L
Tris-HCl,
by successive
washes
in 95°C
150
pH 7.5.
examined.
Results and efprobes.
of segments
stomach,
duodenum,
that each isoactin-specific RNA (mRNA) weight.
molecular
staining
tcRNA
of the
were loaded
not shown). malized
Northern
after
hybridization quantities
respectively
blots were quantitated discussed.’
comparisons
be made
bromide
and methylene
that equivalent
and transferred,
as previously
quantitative could
membrane
used to verify
hybrid-
species of the
Ethidium
of the RNA gel before blotting
were both
of isoactin
of (data
and nor-
In this manner,
for all six actin
ce-
these studies probe
ized to a single messenger
rat
ileum,
and anus confirmed
appropriate
349
the adult
jejunum,
and showed
blue
GI TRACT
from
rectum,
semi-
gene expression
isoforms
examined
in
this study. Northern smooth
blot analysis
muscle
trointestinal tively.
isoactin
of y-smooth expression
tract is shown
Quantitative
expression
analysis
(Figure
muscle
muscle
isoactin
and the transverse
colon
of smooth
coexpressed tract.
muscle
segment
relatively
low levels
isoactin
1 and
bowel,
and rectum. observed
muscle
isoactin;
however,
nately
reduced.
regulated
This muscle
throughout
This
in the
pattern
is
for the y-smooth
the overall
of the smooth
peaks in
the ascending
of expression
to that
is greatly
1
and rectum.
identical
expression
segment
small bowel,
almost pression
of the
low levels of
rat stomach small
Expression
relatively
of the a-smooth
with
throughout
peaks in stomach with
in the esophagus,
Expression
2A and El, respec-
2C) shows that the smooth
rat gastrointestinal
the adult
in the adult rat gasisoactin
the adult
expression
and a-
muscle
are heterogeneously
y-smooth
muscle
in Figure
isoactins
esophagus,
All blots were exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY) using intensifying screens. Each Northern blot series was quantitated using an LKB Ultrascan XL Enhanced Laser Densitometer and the Gelscan XL program (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). Multiple exposures were chosen for each Northern blot series to ensure that each lane was scanned in the established linear range for these studies. Standard Gelscan XL program parameters were established for the initial series of quantitation and maintained for the remainder of the blots analyzed. The quantitation values reported for each lane generally represent an average of at least two different scannings of the same blot to eliminate a variety of blot and/or band inconsistencies. The various quantitation values obtained for each of the four isoactins examined were normalized to a specific activity of 2.8 X 10” cpm . I.tg-’ 0 mL-’ and an exposure time of 96.5 hours. In this way, a relative comparison of the expression of all six actin isoforms could be made for the various bowel segments
ficacy
esophagus,
IN RAT
cum, colon,
colon
Densitometry
Prior studies have shown the specificity of the various isoactin-specific cDNA
blot analysis
staining
Total cellular RNA was isolated from 1.2 g of each specific gastrointestinal segment according to the methods of Chirgwin et a1.i5 For each sample, specific segments were pooled from multiple animals to minimize any animal-specific differences in expression. Total cellular RNA was quantitated spectrophotometrically, and these values were visually confirmed by agarose-formaldehyde gel electrophoresis in the presence of 100 pg/rnL ethidium bromide. In addition, equivalent transfer of total cellular RNA (tcRNA) was verified by methylene blue staining16 of the Northern blot after hybridization. Northern blot analysis was performed with 40 /.tg of total cellular RNA by the method of Thomas” using 2% agarose-formaldehyde gels and Biotrans nylon membranes (ICN Pharmaceuticals Inc, East Hills, NY). Two complete Northern blot series were run on a single gel and blotted to a single membrane to eliminate gel and blotting discrepancies between each Northern blot. Prehybridization, hybridization, and wash conditions were identical to those previously discussed” and were the same for each of the various probes used. When necessary, Northern blots
Northern
EXPRESSION
magnitude
result
of ex-
suggests
isoactins
that
is coordi-
the adult rat gastrointesti-
nal tract. Northern
blot
analysis
3A) and y-cytoplasmic indicates neously
that
of ecytoplasmic
(Figure
the cytoplasmic
coexpressed
tract. Quantitative
3B) isoactin isoactins
in the adult analysis
shows that P_cytoplasmic stomach
segment
1, jejunum,
pression
of the y-cytoplasmic
(Figure isoactin
(Figure expression
are heteroge-
rat gastrointestinal 3C) of these results expression
and ascending isoactin
peaks in colon.
Ex-
peaks in stom-
ach segment 2 and the ascending colon. This pattern is similar to that observed for the P-cytoplasmic isoactin, although the overall magnitude is significantly less and the peak in P-cytoplasmic isoactin expression observed in the adult rat jejunum is not as apparent for the y-cytoplasmic isoactin. In general, the pattern of cytoplasmic isoactin expression appears similar to that observed for the smooth muscle isoactins. These re-
350
LIDDELL ET AL.
GASTROENTEROLOGY Vol. 105, No. 2
A
El
E2
E3 Sl
S2
S3 DU JE
El
E2
E3 Sl
S2
S3 DU JE IL
El
EZ
W
S,
S2
s3
Cu
JE
IL
CE
AC
TC
IL
a,
R
CE AC TC DC RE AN
CE AC TC DC RE AN
AN
SAMPLES
sults suggest that expression tins
is coordinately
well as the smooth Northern 4A)
and
of the cytoplasmic
regulated muscle
blot analysis the a-cardiac
between
isoac-
themselves
as
isoactins. with (Figure
the a-skeletal 4B) isoactins
expression only
shows
that expression of the striated muscle isoactins in the adult rat gastrointestinal tract is primarily limited to segments of the esophagus and colon. Quantitative analysis (Figure 4C) of striated muscle isoactin expression in the adult rat gastrointestinal tract shows that expression of the a-skeletal isoactin peaks in esophagus segment 2 and the cecum and ascending colon of the large bowel. Expression of the cr.-skeletal isoactin decreases dramatically in stomach segments 1 and 2 to reach undetectable levels in stomach segment 3 and the small bowel. Moderate levels of cl-skeletal isoactin
reappear
to decrease
verse colon, (Figure
Figure 2. Northern blot analysis of y-smooth muscle isoactin (A) and a-smooth muscle isoactin (5) expression using 40 ug of total cellular RNA isolated from adult rat gastrointestinal segments including esophagus l-3 (E 1-E3), stomach l-3 (s lS3), duodenum (Du), jejunum (JE), ileum (IL), cecum, ascending colon (AC), transverse colon (TC), descending colon (DC), rectum (RE), and anus (AN). The position of the 18s and 28s ribosomal standards were marked on the original blots. (C) Relative expression of the y-smooth muscle isoactin (0) and a-smooth muscle isoactin (A) isoactins in the adult rat gastrointestinal tract. Quantitation was performed and normalized as discussed in Materials and Methods.
in the cecum
to relatively
descending
levels of a-skeletal
colon,
isoactin
and ascending
low levels
and rectum.
expression
colon
in the transModerate
are also seen in
the anus. Similar patterns of expression are observed for the a-cardiac isoactin with slight peaks in expression occurring in the esophagus and large bowel. These peaks in a-cardiac isoactin expression are much less distinct than those observed for the a-skeletal isoactin, and the overall magnitude of a-cardiac isoactin expression is significantly less than that observed for the a-skeletal isoactin. These results indicate that the striated muscle isoactins are coexpressed within the various bowel segments examined in this study and suggest that these actin isoforms are coordinately regulated in the adult rat gastrointestinal tract. The two
ISOACTIN
August 1993
EXPRESSION
IN RAT
GI TRACT
351
A
El
E2
E3 Sl
S2
S3 DU JE IL
CE AC TC DC RE AN
El
E2
E3 Sl
S2
S3 DU JE IL
CE AC TC DC RE AN
c Figure 3. Northern blot analysis of &cytoplasmic isoactin (A) and y-cytoplasmic isoactin (6) expression using 40 pg of total cellular RNA isolated from adult rat gastrointestinal segments including esophagus l-3 (El-E3),stomach l-3(sl-s3),duodenum (DU), jejunum (JE), ileum (IL), cecum (CE), ascending colon (AC), transverse colon (TC). descending colon (DC), rectum (RE), and anus (AN). The position of the 18s and 28s ribosomal standards were marked on the original blots. (C) Relative expression of the j3-cytoplasmic isoactin (0) and y-cytoplasmic isoactin (A) in the adult rat gastrointestinal tract. The y-cytoplasmic isoactin expression is multiplied by IO. Quantitation was performed and normalized as discussed in Materials and Methods.
peaks in striated tinct
from
cytoplasmic cle isoactins
muscle
isoactin
those observed isoactins,
ulatory
; i
e 5 40 g
20
O S*MPLES
appear
dis-
basis. This observation
muscle
and
more distinct
patterns
that the striated
mus-
ent at the tissue-specific
regulated
these
bowel
the importance
in the regulation
18-23 An analysis
H SC
expression
are independently
mechanisms
expression.
*(
for the smooth
suggesting
other actin isoforms. Prior studies have shown
‘O’
from
of autoreg-
of isoactin
of the y-smooth
gene
muscle/
segments
does not preclude of autoregulation
the fact that may be appar-
level for each of the various
examined.
Discussion Normal the integration
gastrointestinal of numerous
development embryonic
requires
processes
in-
a-smooth muscle ratio, the P-cytoplasmic/y-cytoplasmic ratio, the smooth muscle/cytoplasmic ratio,
volving a variety of developmental paradigms. In an effort to begin to understand these complex processes,
the cl-skeletal/a-cardiac ratio, and the total mRNA content of the various bowel segments
we examined isoactin gene expression in sequential segments of the adult rat gastrointestinal tract. Northern blot analysis of isoactin gene expression in the adult rat gastrointestinal tract shows distinct patterns of smooth muscle, cytoplasmic, and striated muscle isoactin expression. The y-smooth muscle isoactin is
isoactin did not
reveal any distinct patterns (Table 1). The wide degree of variation observed for these ratios suggests that the adult rat gastrointestinal tract independently regulates isoactin gene expression on a segment by segment
352
LIDDELL
ET AL.
El
El
GASTROENTEROLOGY
rat gastrointestinal
No. 2
E2 E3 Sl S2 S3 DU JE IL CE AC TC DC RE AN
E2
E3 Sl
S2
S3 DU JE
IL
CE AC TC DC RE AN
Figure 4. Northern blot analysis of o-skeletal isoactin (A) and a-cardiac isoactin (3) expression using40 pg of total cellular RNA isolated from adult rat gastrointestinal segments including esophagus l-3 (E 1 -E3), stomach l-3 (S 1 -S3), duodenum (DU), jejunum (JE), ileum (IL), cecum (CE), ascending COlOn (AC), VZVISverse colon (TC), descending colon (DC), rectum (RE), and anus (AN). The position of the 18s and 28s ribosomal standards were marked on the original blots. (C) Relative expression of the a-skeletal isoactin (0) and a-cardiac isoactin (A) in the adult rat gastrointestinal tract. Both the a-skeletal isoactin and a-cardiac isoactin expression are multiplied by 10. Quantitation was performed and normalized as discussed in Materials and Methods.
SAMPLES
the predominant
Vol. 105,
actin isoform expressed in the adult tract. The exceptions
to this obser-
striated muscle isoactins in the adult rat gastrointestinal tract such that each pair of these actin isoforms is
vation are the first two segments of the esophagus and
coexpressed
the jejunum where the a-skeletal
consistent
and P-cytoplasmic
in a similar
manner.
These
results are
with prior studies,‘*24*25 which have shown
isoactins predominate, respectively. By comparison, moderate to low levels of a-smooth muscle and P-cy-
that the various actin isoforms are coordinately coexpressed as distinct pairs in most tissues. The present
toplasmic isoactin expression and low to undetectable levels of a-skeletal, y-cytoplasmic, and a-cardiac isoactin expression are also detected in the adult rat
study also shows that coordinate patterns of regulation are observed between the smooth muscle and cytoplasmic isoactins, suggesting that common regulatory pathways may exist for these four actin isoforms. These observations provide additional evidence to support our prior hypothesis that the cytoplasmic isoactins play a significant role in smooth muscle differentiation, maturation, and function.’ In contrast, expression of the striated muscle isoactins appears dis-
gastrointestinal tract. These observations are consistent with our prior developmental studies and show the complex nature of isoactin gene expression in the adult rat gastrointestinal tract. Coordinate patterns of isoactin gene expression are observed for the smooth muscle, cytoplasmic, and
ISOACTIN
August 1993
Table 1. lsoactin mRNA Ratios in Bowel Segments Adult Rat Gastrointestinal Tract
1:l 2:1 9:1 13:l 17:l 21:1 7:1 0.5: 1 13:l 1l:l 4:1 9:1 7:1
48: 1 45: 1 45: 1 15:l
20: 1 25: 1 5:1 11:l
19 25 68 694 408 199 13 32 125 147 441 433 69
6:1 13:l
13:1
31 165
5:l 7:1 3:l
18:l 15:l 23: 1 32: 1 1l:l 28: 1 1O:l 43: 1 17:l 38:1 44:1 38:1 25: 1
2:1 3:1
33: 1 60: 1
a-SM
El E2 E3 Sl s2 s3 DU JE IL CE AC TC DC
0.5: 1 0.5: I 3:1 5:1 1l:l 1O:l 2:1 1:l 20: 1 8:1
RE AN
Total actin
SM/CYT
Y-W
gastrointestinal
a-SKI/ a-CAR
P-CYT/ y-CYT
Samples
of the
-
NOTE. Relative y-smooth muscle (y-SM), a-smooth muscle (a-SM), P-cytoplasmic (P-CYT), y-cytoplasmic (y-CYT). a-skeletal (a-SKL), and a-cardiac (a-CAR) isoactin expression in esophagus segment 1 (El), esophagus segment 2 (E2), esophagus segment 3 (E3), stomach segment 1 (Sl), stomach segment 2 (S2), stomach segment 3 (S3), duodenum (DU), jejunum (JE), ileum (IL), cecum (CE), ascending colon (AC), transverse colon (TC). descending colon (DC), rectum (RE), and anus (AN) of the adult Sprague-Dawley rat. Relative isoactin gene expression was quantitated and normalized as discussed in Materials and Methods. Ratios were rounded to the nearest whole number. CYT equates the sum of the normalized P-cytoplasmic and y-cytoplasmic isoactin expression. SM equates the sum of the normalized y-smooth muscle and a-smooth muscle isoactin expression. Total actin equates the sum of the normalized expression of all six actin isoforms in each bowel segment.
EXPRESSION
tract.
various
caveats
smooth
muscle
This
that
exist
isoactins
terns of smooth
of the
various
Similar
patterns
pression been
stripped
isoactins
observed
The
to cytoplasmic to establish
with
supported McHugh
these
isoactins,
either
the smooth
indicating
muscle
that independent
ways exist for these actin isoforms. autoregulation observed
in the adult
importance the
for the various
mented.‘8-23 most
by recent
significant
has
been
The of
Schevzov
functional cultured
these mechanisms
in whole
organ
it is also possible
that autoregulation
segments.
However,
does not play a
the expression
of the
sphincteric
(phasic)
The P-cytoplasmic adult rat gastrointestinal
in assessing
in isoactin smooth
gastrointestinal
asso-
hypothesis
is
(K. M. that have
gene expres(tonic)
and
muscle
tis-
tract. In ad-
myoblasts.
path-
rat gastrointestinal
This
et a1.23 recently reported distinct for the cytoplasmic isoactins in
autoregulatory difficulty
differences
functions
differences
heterogeneously
in the adult
in modulating
tract.
dition,
Such
in our laboratory
distinct
sues of the adult opossum
docu-
the
role
of
are not
muscle
smooth
in preparation)
nonsphincteric
clearly
reflects
significant
patterns
isoforms
gene expression muscle
segments.
differences
to
and
isoactins.
studies
of
segment
of y-smooth
the differential
et al., manuscript
flanking
path-
in the expression
The fact that similar likely
Distinct
rat gastrointestinal
isoactins
ways are not apparent tract
regulatory
actin
of such mechanisms
cytoplasmic
or cytoplasmic
ratios
bowel
patterns
by the fact that
of isoactin
isoactins
isoactins
and
that the composition
is supported
patterns muscle
muscle
in gastrointestinal
may vary from
in segment-specific
may help ciated
indicating
segment-specific
This observation
to a-smooth
exhave
tract is to establish
apparatus
machinery
these differential
gene that
smooth
of the
in this study suggest
segment.
examined.
isoactin mucosa,
role
cells.
the contractile
in
tissues
to the smooth musbowel each of these individual
primary
muscle
muscle
overlying
the contractile
smooth
in
are intrinsic
in the gastrointestinal
maintain
result
muscle
pat-
differences
segments
within
The
of the
observed
distinct
in bowel
of their
cle tissues found segments.
expression
segments
of smooth
that these differences
the
expression
in the smooth
are observed
makes
cell types irrelevant
gastrointestinal
sion in the functionally from
isoactin
gene expression
353
the segment-specific
tract reflect
isoactin
shown tinct
for the
in other
muscle
GI TRACT
observation
for this study. Consequently, the gastrointestinal
IN RAT
and y-cytoplasmic
coexpressed
isoactins
in all cell types
tract, including
smooth
are
of the mus-
cle cells where they may comprise as much as 30% 40% of the total isoactin mRNA pool.’ The ubiquitous nature
of these actin
isoforms
terpret
the
isoactin
gene expression
segment-specific
makes patterns
observed
it difficult
to in-
of cytoplasmic
in the adult rat gas-
various actin isoforms in the adult rat gastrointestinal tract. The y-smooth muscle and cl-smooth muscle isoactins are heterogeneously coexpressed in all of the
trointestinal tract. However, expression of the cytoplasmic isoactins appears to peak in those bowel segments possessing highly modified epithelia that would require complex cytoskeletal organization. This obser-
bowel segments examined in this study. In situ analysis (K. M. McHugh, manuscript in preparation) has shown that the smooth muscle isoactins are primarily coexpressed in the smooth muscle cells of the mature
vation is consistent with prior studies that have characterized the expression of the cytoplasmic isoactins in the mucosa of various bowel segments.26-28 The predominant cytoplasmic isoactin expressed in the adult
354
LIDDELL
ET AL.
GASTROENTEROLOGY
rat gastrointestinal
tract is the P-cytoplasmic
This observation mental
studies
isoactin form
is consistent that
with our earlier develop-
indicated
that
was the predominant
expressed
cytoplasmic
in most
isoactin
embryonic
gested a functional strating
alterations
of either
study. Therefore,
toplasmic
isoactin
differentiation types found The a-skeletal
of the
cum, colon,
rectum,
tectable
in the poly(A)+
testinal
segments.
nificantly Prior
studies”,12 skeletal
of the adult primarily
striated
muscle
similar
manner,
sphincteric this
would
skeletal
for
in these bowel muscle
a source
for
the
on the normal
and function isoforms
occurs
type(s)
found
tract,
that
some
within
the
suggesting
striated
muscle
Whether
isoactins
the
with
expression
an obvi-
detection segments.
of In a
in the
detection
histologically argue pressed
striated muscle isoactin in this bowel segment. Similar sources of skeletal muscle are not, however, known to exist in the cecum, colon, or rectum of the adult rat. More detailed histological analysis of these segments is currently underway in an attempt to clarify these issues. As with the smooth muscle isoactins, the primary function of the striated muscle isoactins appears to involve the establishment and maintenance of the contractile apparatus found in skeletal and cardiac muscle. It is postulated that the a-skeletal and a-car-
striated
studies
at the protein distinct
and
differentially
nature
muscle
expression
have
characterized muscle
the
isoactin
The identification
muscle
isoactins muscle
of
in the esophagus,
rat seems
to strongly
are being
level in these tissues. skeletal
of these
are not available.
of the adult muscle
in
level
of the striated skeletal
of our reisoactins
of the adult rat, simi-
and the heart.
that the striated
organs
level in the adult rat gastroinof the novel segments
distinct
protein
that the cytoplaswere
the differential
and anus
histologically of
isoactin
showed
at the protein
muscle
of
to be determined.
isoactins
of the
expression
in skeletal stomach,
Because
numerous
differential
patterns
of gastrointestinal
muscle
verifying
pat-
in the adult
in identical
remains
clearly
at the protein
However,
role for the
of this study are very consistent
studies
gastrointestinal
Skel-
gastrointestinal
observed
smooth
isoforms
cell
in these tissues.
in a variety
lar studies
actin
muscle
segment-specific
various actin
rat
studies 29-32 characterizing
These
of these
heterogeneous
the results
expression tissues.
tract. However,
functional
tract result
protein
prior
adult
the
development
gene expression
rat gastrointestinal However,
of skeletal to clarify
nonstriated
an expanded
the
that the rat cecum,
the expression
in
of the then
small quantities
of the rat gastrointestinal
it is also possible
detection
tissues of the anus. As with the esophagus, provide
muscle
ported
zone be-
is also present
be necessary
tract.
in stomach
provide the
therefore
role of this skeletal
indicate
smooth
hypotheses
as absolute,
It would
expressed
and
is taken
muscle
the
specificity
contain
testinal
analysis
of these
of this study would
studies.
its extent. found
isoactins
and sigof
both
and rectum
mic and
that it is composed
in the transition
muscle
the
expression
Histological
and stomach
isoactins
that
to the developing
muscle throughout
of skeletal
(data
from
If the tissue
No. 2
differences.
striated
results
isoactin ce-
is also de-
confirm
1 and 2. These observations
source
verified
that
shows
is even detected
studies
developmental
and heart.
rat esophagus
the
of these same gastroin-
is restricted
the esophagus
segments ous
stomach,
isoactins
suggested
muscle
of skeletal
etal muscle tween
earlier
have
these actin isoforms mature
have
muscle
fraction
our
that
the
distinct
However,
terns of isoactin
are heterogeneously
These observations
extend
cy-
of the cell
show
and anus. Additional
of the striated
in the
tract.
rat esophagus,
in our laboratory
expression
study
isoactin
are ob-
functional that
colon, muscle.
in the normal
of many
present
in the adult
muscle
of a precise
rat gastrointestinal
and a-cardiac
striated
examined
ratio may be essential
coexpressed not shown)
segments
and development
results
during
This hy-
ratios
maintenance
in the adult
to be verified.
that distinct
isoactin
served for most of the bowel
isoactins.
sug-
distinct
are functionally
muscle
have
morphology.
possess
it is assumed
remain
cells results
by the observation
isoactins addition,
the y-
the P-cytoplas-
in cultured
in cellular
P-cytoplasmic/y-cytoplasmic present
studies23
In
iso-
for this data by demon-
isoactin
is supported
whereas
diac
isoactins
actin
expressed
Recent
significance
mic and y-cytoplasmic in distinct
tissues,
was primarily
that overexpression
pothesis
the bcytoplasmic
cytoplasmic
adult
development.
isoactin.
Vol. 105.
ex-
In contrast,
has never
been
reported in the cecum, colon, or rectum of the adult rat. A histological review by our laboratory of random colonic segments from the rat, human, dog, and guinea pig is consistent with these results in not identifying any distinct skeletal muscle fibers within these organ segments (unpublished observation). Consequently, even though a-skeletal and a-cardiac isoactin mRNA is detected in these bowel segments, it is possible that the striated muscle isoactin proteins are not expressed. Similar types of ectopic expression of a variety of apparently tissue-specific transcripts have been reported studies are in several studies. 33,34 Clearly, additional
August
ISOACTIN
1993
needed to further characterize the precise nature of this expression in the adult rat gastrointestinal tract. In summary, the results of the present study show that the actin multigene family is heterogeneously coexpressed in a segment-specific manner in the adult rat gastrointestinal tract. In addition, distinct patterns of striated muscle isoactin gene expression are also observed in specified segments of the adult rat gastrointestinal tract. Both coordinate and independent regulation of isoactin gene expression is observed in the gastrointestinal tract, although distinct patterns of autoregulation are absent. These observations indicate that the mature gastrointestinal tract of the rat is highly complex and capable of segment-specific regulation of isoactin gene expression using a variety of molecular mechanisms. Whether this type of local regulation of gene expression results in, or is the product of, the distinct functional differences observed for the various bowel segments of the adult rat gastrointestinal tract remains to be determined. In conclusion, this study provides a basis for future studies designed to investigate the factors controlling the normal development and resulting diversity of the adult rat gastrointestinal tract.
tal expression
EXPRESSION
IN RAT
GI TRACT
of a smooth muscle y-actin in postmeiotic
355
male
germ cells of mice. 1989;9: 1875- 188 1.
10. Gunning P, Ponte P, Blau H, Kedes L. a-Skeletal and a-cardiac actin genes are coexpressed in adult human skeletal muscle and heart. Mol Cell Biol 1983;3: 1985- 1995.
11. Alonzo S, Garner I, Vandekerckhove
J, Buckingham
M. Genetic
analysis of the interaction between cardiac and skeletal actin gene expression in striated muscle of the mouse. J Mol Biol 1990;2 11:727-738. 12. McHugh K, Lessard J. The developmental expression of the rat a-vascular and y-enteric smooth muscle isoactins: isolation and characterization
of a rat y-enteric
1988;8:5224-523
actin cDNA. Mol Cell Biol
1.
13. Erba H, Gunning P, Kedes L. Nucleotide sequence of the human y-cytoskeletal actin mRNA: anomalous evolution of vertebrate non-muscle actin genes. Nucleic Acids Res 1986; 14:52755294. 14. Gunning P, Ponte P, Okayama H, Engel S, Blau H, Kedes L. Isolation and characterization
of full-length cDNA clones for human a-,
p-, and y-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is sequentially removed. Mol Cell Biol 1983;3:787-795. 15. Chirgwin J, Pryzbla A, MacDonald R, Rutter W. Isolation of biologically active ribonucleotide acid from sources enriched in ribonucleases. Biochemistry 1979; 18:5294-5299. 16. Maniatis T, Fritsch E, Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982;206. 17. Thomas P. Hybridization of denatured ments
transfered
to nitrocellulose.
RNA and small DNA fragProc Natl Acad Sci USA
1980;77:5201-5205.
References
18. Leavitt J, Ng S, Aebi U, Varma M, Latter G, Burbeck S, Kedes L, Gunning P. Expression oftransfected mutant p-actin genes: alterations of cell morphology and evidence for autoregulation in actin pools. Mol Cell Biol 1987;7:2457-2466.
1. McHugh K, Crawford K, Lessard J. A comprehensive
analysis of expression of the isoactin
the developmental and tissue-specific multigene family in the rat. Dev Biol 1991; 148:442-458. 2.
Erba H, Eddy R, Shows T, Kedes L, Gunning L. Structure, chromosome location, and expression of the human y-actin gene: differential evolution, location, and expression of the cytoskeletal 8and y-actin genes. Mol Cell Biol 1988;8: 1775- 1789.
3.
Tokunaga K, Takeda K, Kamiyama K, Kageyama H, Takenaga K, Sakiyama S. Isolation of cDNA clones for mouse cytoskeletal y-actin and differential expression of cytoskeletal actin mRNAs in mouse cells. 1988;8:3929-3933.
4.
Kapanci Y, Burgan S, Pietra G, Conne B, Gabbiani G. Modulation of actin isoform expression in alveolar myofibroblasts (contractile interstitial cells) during pulmonary hypertension. Am J Pathol 1990; 13688 l-889.
5.
Leslie K, Mitchell J, Woodcock-Mitchell J, Low R. Alpha smooth muscle actin expression in developing and adult human lung. Differentiation 1990;44: 143-149.
6.
Ramadori G, Schwogler S, Dienes H, Knittel T, Rieder H, Meyer zum Buschenfelds K. Expression of the gene of the u-smooth muscle-actin isoform in rat liver fat-storing (ITO) Cells. Virchows Arch B Cell Pathol 1990;59:349-357.
7.
Ruzicka D, Schwartz R. Sequential activation of a-actin genes during avian cardiogenesis: vascular smooth muscle a-actin gene transcripts mark the onset of cardiomyocyte differentiation. J Cell Biol 1988; 107:2575-2586.
19. Ng S, Erba H, Latter G, Kedes L, Leavitt J. Modulation of microfilament protein composition by transfected genes, Mol Cell Biol 1988;8: 1790- 1794.
cytoskeletal
actin
20. Gunning P, Gordon M, Wade R, Gahlmann R, Lin C-S, Hardeman E. Differential control of tropomyosin
mRNA levels during myo-
genesis suggests the existence of an isoform competition-autoregulatory compensation control mechanism. Dev Biol 1990; 138:443-453. 21.
Wade R, Sutherland C, Reinhold G, Kedes L, Hardeman E, Gunning P. Regulation of contractile protein gene family mRNA pool sizes during myogenesis. Dev Biol 1990; 142:270-282.
22.
Llyod C, Schevzov G, Gunning P. Transfection
of nonmuscle
p
and y-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes. J Cell Biol 1992; 117:787-797. 23.
24.
Schevzov G, Catriona L, Gunning P. High level expression of transfected 6- and y-actin genes differentially impacts on myoblast cytoarchitecture. J Cell Biol 1992; 1 17:775-785. Gunning P, Ponte P, Blau H, Kedes L. a-Skeletal and a-cardiac actin genes are coexpressed in adult human skeletal muscle and heart. Mol Cell Biol 1983;3: 1985- 1995.
25.
Hayward L, Schwartz R. Sequential expression of chicken actin genes during myogenesis. J Cell Biol 1986: 102: 1485- 1493. Drenckhahn D, Dermietzel R. Organization of the actin filament cytoskeleton in the intestinal brush border: quantitative and qualitative immunelectron microscope study. J Cell Biol 1988;107:
8.
Sawtell N, Lessard J. Cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the a-vascular but not the y-enteric isoform in differentiating striated myocytes. J Cell Biol 1989; 109:2929-2937.
26.
9.
Kim E, Waters S, Hake L, Hecht N. Identification and developmen-
27. Cheng H, Bjerknes M. Asymmetric distribution of actin mRNA and
1037-1048.
356
28.
29.
30.
31. 32.
LIDDELL
ET AL.
cytoskeletal pattern generation in polarized epithelial cells. J Mol Biol 1989;210:541-549. Weiser M, Sykes D, Killen P. Rat intestinal basement membrane synthesis. Epithelial versus nonepithelial contributions. Lab Invest 1990;62:325-330. Vandekerckhove J, Weber K. At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptide. J Mol Biol 1978; 122:783-802. Vandekerckhove J, Weber K. The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. Differentiation 1979;14:123-133. Fatigati V, Murphy R. Actin and tropomyosin variants in smooth muscles. J Biol Chem 1984;259: 14383-14388. Kedinger M, Simon-Assman P, Bouziges F, Arnold C, Alexandre E, Haffen K. Smooth muscle actin expression during rat gut devel-
GASTROENTEROLOGY
Vol. 105.
No. 2
opment and induction in fetal skin fibroblastic cells associated with intestinal embryonic epithelium. Differentiation 1990;43: 87-97. 33. Sarkar G, Sommer 5 Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. Science 1989;244:33 l-334. 34. Chelly J, Concordet J-P, Kaplan J-P, Kahn A. Illegitimate transcription: transcription of any gene in any cell type. Biochemistry 1989;86:2617-262 1.
Received August 18, 1992. Accepted April 5, 1993. Address requests for reprints to: Kirk M. McHugh, Ph.D., Thomas Jefferson University, 1020 Locust Street, Philadelphia, Pennsylvania 19107. Supported by National Institutes of Health grant HD27252. The authors thank J. Brittingham for assistance In this project.