© 1998 American Heart Association, Inc.
Rapid Communications |
Smooth Muscle–Specific Expression of the Smooth Muscle Myosin Heavy Chain Gene in Transgenic Mice Requires 5'-Flanking and First Intronic DNA Sequence
From the Department of Molecular Physiology and Biological Physics (C.S.M., C.P.R., J.E.H., I.M., G.K.O.), University of Virginia, Charlottesville; Cardiovascular Drug Discovery (C.S.M.), Bristol-Myers Squibb, Princeton, NJ; and the Department of Molecular Physiology and Biophysics (S.L.W.), University of Vermont, Burlington.
Correspondence to Gary K. Owens, PhD, Box 449 Health Sciences Center, University of Virginia School of Medicine, Charlottesville, VA 22908. E-mail gko{at}virginia.edu
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Abstract—The smooth muscle myosin heavy chain (SM-MHC) gene encodes a major contractile protein whose expression exclusively marks the smooth muscle cell (SMC) lineage. To better understand smooth muscle differentiation at the transcriptional level, we have initiated studies to identify those DNA sequences critical for expression of the SM-MHC gene. Here we report the identification of an SM-MHC promoter-intronic DNA fragment that directs smooth muscle–specific expression in transgenic mice. Transgenic mice harboring an SM-MHC-lacZ reporter construct containing
16 kb of the SM-MHC genomic region from -4.2 to +11.6 kb
(within the first intron) expressed the lacZ
transgene in all smooth muscle tissue types. The inclusion of the
intronic sequence was required for transgene expression, since 4.2 kb
of the 5'-flanking region alone was not sufficient for expression. In
the adult mouse, transgene expression was observed in both
arterial and venous smooth muscle, in airway smooth muscle
of the trachea and bronchi, and in the smooth muscle layers of all
abdominal organs, including the stomach, intestine, ureters, and
bladder. During development, transgene expression was first detected in
airway SMCs at embryonic day 12.5 and in vascular and visceral SMC
tissues by embryonic day 14.5. Of interest, expression of the SM-MHC
transgene was markedly reduced or absent in some SMC tissues, including
the pulmonary circulation. Moreover, the transgene exhibited a
heterogeneous pattern between individual SMCs within a
given tissue, suggesting the possibility of the existence of different
SM-MHC gene regulatory programs between SMC subpopulations and/or of
episodic rather than continuous expression of the SM-MHC gene. To our
knowledge, results of these studies are the first to identify a
promoter region that confers complete SMC specificity in vivo, thus
providing a system with which to define SMC-specific transcriptional
regulatory mechanisms and to design vectors for SMC-specific gene
targeting.
Key Words: smooth muscle differentiation • myosin heavy chain • gene targeting • smooth muscle–specific expression
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Introduction |
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Our understanding of muscle differentiation has been greatly enhanced in recent years with the identification of several key cis elements and trans factors that regulate expression of muscle-specific genes.1 2 However, the elucidation of transcriptional pathways that govern muscle differentiation has been primarily restricted to skeletal and cardiac muscle. To date, no transcription factors have yet been identified that direct SM-specific gene expression, or SMC myogenesis.3 Unlike skeletal and cardiac myocytes, the SMC does not undergo terminal differentiation and exhibits a high degree of phenotypic plasticity, both in culture and within the animal.3 4 Phenotypic plasticity is particularly striking when SMCs located in the media of normal vessels are compared with SMCs located in intimal lesions resulting from vascular injury or atherosclerotic disease.4 5 6 7 Major modifications include decreased expression of SM isoforms of contractile proteins, altered growth regulatory properties, increased matrix production, abnormal lipid metabolism, and decreased contractility.3 The process by which SMCs undergo such changes is referred to as "phenotypic modulation."8 Importantly, these alterations in SMC protein expression patterns cannot be viewed simply as a consequence of vascular disease; instead, they are likely to contribute to its progression.
A key to understanding SMC differentiation is to identify
transcriptional mechanisms that control expression of genes that are
selective or specific for differentiated SMCs and that are required for
its principal differentiated function, contraction. To this end, our
laboratory and others have studied the expression of the contractile
proteins SM 
-actin9 10 and
SM-MHC,11 12 13 14 15 16 as well as a variety of proteins
implicated in control of contraction, including
SM22,17 18
h1-calponin,19
h-caldesmon,20 telokin,21
and desmin.22 Of these gene products, only
SM-MHC expression appears to be completely restricted to SMC lineages
throughout development.23 All others show at
least transient expression in non-SMC tissues.3
As such, at present, the SM-MHC gene is unique with regard to its
potential utility for identification of SMC-specific transcriptional
regulatory pathways and mechanisms. To date, four SM-MHC isoforms
(SMC-1A, SMC-1B, SMC-2A, and SMC-2B) have been
identified,24 25 26 all of which are derived from
alternative splicing of a single gene.23 27
Alterations in expression of SM-MHC isoforms have been extensively
documented in SMCs that have undergone phenotypic modulation either
when placed in culture28 29 or in vascular
lesions of both humans and several animal models of vascular
disease.30 31 Thus, the SM-MHC gene
represents an excellent candidate gene for delineating
transcriptional pathways important for both normal development and
diseased states.
Transcriptional regulation of the SM-MHC gene has been analyzed extensively in cultured SMCs, and several functional cis elements have been identified.11 12 13 14 15 16 However, because differentiation of SMCs is known to be dependent on many local environmental cues that cannot be completely reproduced in vitro, cultured SMCs are known to be phenotypically modulated compared with their in vivo counterparts.3 8 As such, certain limitations may apply regarding the usefulness of cultured SMCs in defining transcriptional programs that occur during normal SMC differentiation and maturation within the animal. Using transgenic mice to identify DNA sequences critical for SM-MHC expression, we have begun to investigate the molecular mechanisms that regulate SMC-specific transcription within the animal during normal development. In the present study, we report for the first time the identification of sufficient regions of the SM-MHC gene to direct SMC-specific expression in vivo in transgenic mice.
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Isolation and Cloning of the Rat SM-MHC Promoter
The SM-MHC gene contains a very short untranslated first exon (88 bp in rat) that is followed by a >20-kb first intron.32 The cloning and sequence of the 5'-flanking region of the rat SM-MHC gene (nucleotides -4220 to +88) have been previously reported.11 15 To obtain the 5'-flanking sequence with additional intronic DNA, we screened a rat genomic phage library (Stratagene Corp) using standard Southern blotting techniques and a 32 P-radiolabeled 45mer oligonucleotide corresponding to the conserved untranslated first exon as a probe (nucleotides +14 to +58). One of the positive recombinant phage
clones identified
contained an 16-kb insert (determined by restriction enzyme
analysis and partial sequencing) that spanned the SM-MHC gene
from nucleotides -4229 to +11 600. Identical
restriction enzyme patterns between rat genomic DNA and multiple
positive clones revealed that none of the clones identified had
undergone rearrangement.
Construction of the Rat SM-MHC-lacZ
Reporters
The pUC19-lacZ vector used in the generation of
SM-MHC reporter gene constructs was kindly provided by Dr Eric
Olson.33 To facilitate removal of pBS plasmid
DNA, the pUC19-lacZ vector was modified by inserting
NotI restriction enzyme recognition sites at the
HindIII and EcoRI sites located at the borders of
the pBS vector sequence. Two SM-MHC-lacZ reporter genes
were constructed for the generation of transgenic mice (Figure 1
). One construct
(p4.2-lacZ) was created by ligating an 4.3-kb
BglII fragment that extended from nucleotides
-4220 to +88 (see Reference 1515 ) into a unique BamHI site of
the pUC19-lacZ vector, and the other construct tested
(p4.2+intron-lacZ) was generated by subcloning an
16-kb SalI fragment that extended from
nucleotides -4229 to +11 600 into the SalI
site of the pUC19-lacZ vector. To facilitate splicing of
the p4.2+intron-lacZ construct, a synthetic splice
acceptor site was ligated into the KpnI site of the
pUC19-lacZ vector before insertion of the SM-MHC DNA
fragment. The location of the KpnI site, between the
SalI site and the lacZ gene, allowed for the
correct positioning of the splice acceptor site at the +11 600 end of
the SM-MHC intron. The proper construction of each
SM-MHC-lacZ chimeric plasmid was verified by sequencing
and restriction enzyme analyses. As an additional precaution
against cloning artifacts, both transgenic constructs were tested for
lacZ expression in transient transfection assays in
cultured rat aortic SMCs using a method that was previously
described.15 In this assay, both constructs were
determined to be positive for lacZ expression.
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Generation and Analysis of Transgenic Mice
Plasmid constructs p4.2-lacZ and
p4.2+intron-lacZ were tested for SM-MHC promoter
activity in transgenic mice after removal of the pBS vector DNA through
NotI digestion and subsequent agarose gel purification.
Transgenic mice were generated using standard
methods,17 34 either commercially (DNX) or within
the Transgenic Core Facility at The University of Virginia,
Charlottesville. Transgenic mice were either killed and
analyzed during embryological development (transient
transgenics) or were used to establish breeding founder lines (stable
transgenics). Transgene presence was assayed by polymerase chain
reaction using genomic DNA purified from either placental tissue
(embryonic mice) or tail clips (adult mice) according to the method of
Vernet et al.35 Transgene expression and
histological analyses were performed as
described previously.17 33
SM-MHC Immunohistochemistry
Various SM-containing tissues were collected from 5- and
6-week-old transgenic mice and fixed overnight in methacarn (60%
methanol, 30% chloroform, and 10% glacial acetic acid). Tissues were
subsequently dehydrated through a graded series of methanol. Fixed
dehydrated tissues were prepared for paraffin embedding by incubation
in 100% xylene. Tissue was then infiltrated by incubation through a
series of xylene:paraffin (3:1, 1:1, and 1:3) solutions and two final
incubations in 100% paraffin before embedding in 100% paraffin.
Serial sections (6 um) were placed on uncoated slides and then dried
for 45 minutes on a slide warmer set at 40°C. Sections were
cleared in multiple washes of 100% xylene and rehydrated through a
graded ethanol series to a final incubation in PBS.
Endogenous peroxidase activity was quenched by incubating
slides in methanol containing 0.3% hydrogen peroxide for 30 minutes.
Slides were subsequently rehydrated in PBS and blocked in a 1:50
solution of normal goat serum made up in PBS. Sections were then
incubated with the primary antibody for 1 hour and washed with three
changes of PBS. Detection of primary antibody was performed using a
Vectastain ABC kit according to the instructions of the manufacturer
with DAB as the chromagen (Vector Laboratories).
Several different SM-MHC antibodies were tested. This included a
monoclonal antibody designated 9A9 that we have previously
characterized36 and that shows reactivity with
the SM-1 and SM-2 isoforms of SM-MHC but shows no reactivity with
nonmuscle MHCs or other proteins. However, whereas this antibody showed
some reactivity with mouse SM-MHC isoforms in Western analyses,
it reacted very poorly with mouse SM-MHCs in fixed tissues. In
addition, although a polyclonal SM-MHC peptide antibody provided by
Nagai et al24 showed complete specificity for
SM-MHC isoforms in Western analyses of SM tissues from multiple
species, this antibody showed little or no reactivity with mouse SM-MHC
isoforms. To circumvent these limitations, we used a rabbit
anti–chicken gizzard SM-MHC polyclonal antibody provided by Dr Ute
Groschel-Stewart (University College London).37
We found that this antibody exhibited strong reactivity and complete
specificity for SM-1 and SM-2 MHC isoforms on the basis of Western
analyses of multiple mouse tissues. Moreover,
consistent with previous reports,37 this
antibody showed complete specificity for SM tissues on the basis of
immunostaining of multiple adult and embryonic tissues
(D. Raines and G.K. Owens, unpublished data, 1998). This rabbit
anti–chicken gizzard SM-MHC polyclonal antibody was used at a
concentration of 20 µg/mL in PBS. Biotinylated goat anti-rabbit
secondary antibodies were purchased from Vector Laboratories and used
at a concentration of 10 µg/mL in PBS. Detection was carried out as
described in the Vectastain kit using DAB as the chromagenic substrate.
Appropriate immunohistological controls were performed
to assess specificity, including exclusion of primary antibody and use
of control nonimmune rabbit serum.
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Results |
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Expression of the SM-MHC-lacZ Reporter Gene in Transgenic Mice
We previously reported that an SM-MHC promoter DNA fragment extending from nucleotides -4220 to + 88 was capable of directing high-level expression in cultured rat aortic SMCs.15 When tested in bovine endothelial cells, L6 myoblasts, and L6 myotubes, the activity of this construct was determined to be negligible. To determine whether this same promoter DNA fragment was capable of directing SMC-specific expression in the animal, this fragment was subcloned into a pBS-lacZ reporter gene construct (p4.2-lacZ) and tested for activity in transgenic mice. Thirteen independent transient transgenic mice harboring the p4.2-lacZ transgene were generated and analyzed for lacZ expression at multiple embryological stages ranging from E13.5 to E19.5. For all transgenic mice, we were unable to detect any transgene expression. These data suggest that in contrast to activity levels observed for cultured SMCs,15 the SM-MHC promoter fragment present within the p4.2-lacZ construct did not contain sufficient DNA required for directing SMC-specific expression in transgenic mice.
Portions of the SM-MHC First Intron Were Required for Directing
SMC-Specific Expression in Transgenic Mice
It is well documented that cis elements important for
gene expression can be found outside the 5'-flanking region, including
intronic regions. Because 4.2 kb of 5'-flanking DNA was found to be
insufficient for expression in vivo, we tested a larger construct with
an added intronic sequence. We screened a rat genomic phage library and
identified one recombinant clone whose insert contained 4229 bp of the
5'-flanking region, 88 bp of the first exon, which is the untranslated
sequence, and an additional 11.5 kb of first intronic sequence (total
span, -4.229 to +11.6). This fragment, which was essentially identical
to the p4.2-lacZ construct with respect to 5'-flanking
sequence and the presence of the 88 bp of the 5'-untranslated sequence,
was isolated from phage by SalI digestion and subcloned
into the pUC19-lacZ vector to create the
SM-MHC–reporter gene plasmid p4.2+intron-lacZ. This
construct was initially tested in cultured SMCs, where it was found to
exhibit an 2- to 3-fold increase in activity compared with
p4.2-lacZ.
The reporter gene p4.2+intron-lacZ was used to generate
four independent transgenic mice: one mouse was killed at E13.5 for
transgene expression analysis, and the other three were
established as stable transgenic founder lines (designated as 2282,
2642, and 2820) that were used for analysis of transgene
expression throughout embryological development and early adulthood.
Analysis of adult mice generated from the three stable founder
lines showed that lacZ transgene expression was
essentially identical between the three founders and completely
restricted to SM (Figure 2). Gross
examination of the heart and lung region excised from a 5-week-old
p4.2+intron-lacZ mouse and assayed for
lacZ expression revealed that transgene expression was
present in the descending thoracic aorta, coronary
arteries, trachea, and bronchi (Figure 2A). Transgene expression was
not detected in any non-SM tissues in this region, such as heart muscle
and lung tissue. Of note, transgene expression was also not detected in
SM-containing tissues in this region, including the esophagus, or in
any of the blood vessels located within the lungs. Transgene
expression was readily detectable in the major branches of the
coronary arterial tree, including the left and
right coronary arteries (Figure 2B), as well as in small
coronary arteries and arterioles (Figure 2D) of 5- to
6-week-old transgenic mice. However, no lacZ expression
could be detected in any of the coronary veins (Figures 2B, 2D, and 3C). Transgene expression was also readily detected in the
descending thoracic aorta and intercostal arteries (Figure 2C) as well
as throughout blood vessels in the extremities and main body trunk,
including many small arteries, arterioles, and veins, such as the
mesentery vessels (Figure 2E). In contrast, expression was absent in
SMCs within all pulmonary vessels, and little expression was
seen in the most proximal regions of the cardiac outflow tracts.
Expression of the lacZ transgene was also readily
detectable in the visceral SM of the intestine (Figure 2F), the ureter
and bladder (Figure 2G), the stomach (Figure 2H), and the uterus and
gallbladder (not shown). Thus, these initial analyses
demonstrated that the p4.2+intron-lacZ construct
contained sufficient DNA for expression in all SMC tissue types,
although certain SMC tissues were negative at least in 5- to 6-week-old
animals. Moreover, certain SM tissues, such as the aorta (Figure 2C),
intercostal arteries (Figure 2C), jejunum (Figure 2F), and stomach
(Figure 2H), clearly showed a mosaic pattern of transgene expression
that was visible even at the gross tissue level.
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To assess transgene expression at the cellular level, we performed a
histological analysis of lacZ
reporter expression (Figure 3
). Results
of these studies further demonstrated that transgene expression was
highly restrictive to SM. For example, analysis of the bladder
(data not shown) and airway SM (Figure 3A) demonstrated that transgene
expression was highly specific and appeared to be present in
virtually all SMCs located within these tissues. In contrast, SMCs
within many SM tissues, including the aorta (Figure 3B),
coronary vessels (Figure 3C), the intestine (Figure 3D),
stomach (not shown), and many smaller blood vessels, including small
arteries, arterioles, veins, and venules (Figure 3E and 3F), displayed
a heterogeneous pattern of expression. Transgene expression
was easily detectable in some cells but appeared to be absent in
immediately adjacent cells. Results of serial sectioning showed that
these observations were not due to analysis of a single
sectioning plane. We also saw evidence for
heterogeneity of transgene reporter expression within
small vessels that lie in proximity within a given tissue. For example,
Figure 3F depicts two arteries and a vein located in the parenchymal
tissue surrounding the small intestine. lacZ expression
is clearly visible in a portion of the vein and in one of the arteries,
yet it is undetectable in the immediately adjacent artery. In contrast
to these observations, analysis of SM-MHC protein expression in
individual SMC by immunostaining with an
SM-MHC–specific antibody showed detectable expression in all SMCs
within these tissues, including the thoracic aorta and jejunum (Figure 4). Taken together, these results
indicate that although the p4.2+intron-lacZ transgene
exhibited SMC-specific activity and was expressed in all major SM
types, it exhibited marked differences in activity in subsets of SMCs
both within and between different adult SMC tissues.
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Transgene Expression in the Developing Embryo
To determine whether expression of the
p4.2+intron-lacZ transgene resembled the developmental
expression pattern of the endogenous SM-MHC gene, embryos
from the three stable founder lines were obtained at various stages
throughout development (E10.5 through E19.5) and analyzed for
lacZ expression. Additionally, one transient founder was
generated and analyzed for transgene expression at E13.5.
Transgene expression patterns were essentially identical in all four
independent transgenic lines (ie, one transient transgenic mouse and
three stable founder lines). Expression of the SM-MHC transgene was
completely restricted to SMCs, although transient expression in the
atrial myocardium was observed between E12.5 and E17.5 in
one of the stable lines (data not shown). However, this presumably was
the result of some insertional effect, since this was not observed in
the remaining three lines nor was expression of the
endogenous gene detectable in the
heart.23 Transgene expression patterns of embryos
derived from stable founder lines 2282, 2642, and 2820 are
presented in Figures 5 and 6. The earliest developmental stage at
which transgene expression could be detected was E12.5, where
lacZ expression was readily identified in the trachea
and bronchi (Figure 5A and 5B). By E14.5, transgene expression was
detectable in the bronchi, intestine, stomach, trachea, and the aorta,
as well as a few other vessels throughout the embryo (Figure 5B). Of
particular interest, although transgene expression was virtually absent
in the esophagus in the adult (Figure 2H), its expression was clearly
evident in embryos (Figure 5A through 5D). At E16.5, transgene
expression was more pronounced in the aorta than at earlier
developmental time points, although it had a variegated and less
intense appearance relative to other SM tissues (Figure 5C).
Additionally, the frequency of vessels that were positive for transgene
expression was higher in peripheral vessels, particularly
those located in the extremities of the animal.
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One of the most notable differences between the E16.5 and E19.5
embryos was a marked increase in the frequency of blood vessels that
stained positive for lacZ expression (Figures 5D and 6).
However, lacZ expression remained undetectable in a
number of vessels. Especially conspicuous was the general absence of
expression in many of the large blood vessels in the head and neck
region, including the internal and external carotid arteries, the
jugular vein, and the cerebral arteries and veins. However, many
smaller-sized blood vessels were positive for transgene expression in
the head and neck region. Transgene expression was also readily
detectable in many other arteries and veins throughout the body,
including the iliac artery and vein (Figure 6B), the caudal artery and
vein, the femoral artery, the umbilical arteries and vein, the ulnar
and radial arteries, and the superficial arterioles and venules within
the musculature of the thoracic cage (Figure 6C).
Although expression levels in these types of studies are certainly not quantitative, it is worth noting that levels of lacZ staining within the aorta did not appear to be as intense as staining within many other blood vessels and visceral SM tissues. In summary, results of these embryological studies support the data gathered from analysis of transgene expression in juvenile and adult mice and indicate that the p4.2+intron-lacZ construct contains sufficient DNA for directing SMC-specific expression in all SMC tissue types. However, results leave open the possibility that additional genomic regions may be required for SM-MHC expression in some subsets of SMCs.
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The SM-MHC lacZ Transgene Was Specifically Expressed in All SMC Tissue Types
To begin to understand the mechanisms that regulate expression of the SM-MHC gene, we used transgenic mice to identify an
16-kb
promoter-intronic fragment (nucleotides -4229 to
+11 600) that was sufficient for directing SMC-specific expression
in vivo. Previously, we as well as others have identified multiple
cis elements contained within the first 4.2 kb of the
5'-flanking sequence of the SM-MHC promoter that are critical for
expression in cultured SMCs.11 12 13 14 15 16 The fact that
the p4.2-lacZ construct was found to be active in
cultured SMCs, but not in the mouse, indicates that additional
regulatory elements are necessary for expression within the in vivo
context. The fact that the p4.2+intron-lacZ construct
was expressed in SMC tissues within the mouse, whereas the
p4.2-lacZ construct was inactive, strongly suggests that
the first 11.5-kb region of intron 1 contains enhancer elements
required for expression in vivo but not in cultured SMCs. Differences
in requirements for expression of the SM-MHC gene in cultured SMCs
versus in vivo SMCs in the mouse may be the result of the generalized
phenotypic modulation of SMCs that occurs in cell
culture3 or may reflect alterations in specific
local environmental cues that differ between in vivo and culture
conditions. For example, the cultured SMCs used in the present
studies are known to constitutively produce active
TGF-ß1.38 39 Moreover, we have previously shown
that TGF-ß1 markedly stimulates SM-MHC expression in our cultured rat
aortic SMCs and have presented evidence suggesting that this
may be mediated by a conserved TGF-ß1 control element found within
the 4.2-kb 5' promoter region.38 Although direct
evidence is lacking, it is possible that the high activity of the
4.2-kb promoter fragment in cultured SMCs may be, at least in part,
TGF-ß1 dependent. There, of course, remain many other possibilities,
including the presence or absence of mechanical forces in the culture
system. Reusch et al40 have shown that cyclic
stretch of cultured aortic SMCs derived from neonatal rats stimulates
SM-MHC expression and that such effects are dependent on specific
cell–extracellular matrix interactions. In any case, much additional
work will be required to define the mechanisms responsible for the
differences in genomic regions required for SM-MHC expression in SMCs
in vivo versus in vitro.
Heterogeneity of Transgene Expression in
Vascular SMCs
There is extensive evidence for functional and structural
heterogeneity of SMCs both between and within different
SMC tissues.41 42 43 This is not surprising given
the plasticity of the SMC and the fact that it must carry out very
diverse functions at different developmental stages and in response to
injury or pathological stimuli.3 8 44 Despite the
clear evidence for heterogeneity among SMC
subpopulations, the underlying mechanisms responsible for phenotypic
diversity are not well understood. Results of the present studies
revealed distinct patterns of transgene expression with respect to
developmental stage and SMC tissue type. For example, we
consistently were unable to detect transgene expression in
certain blood vessels, including the pulmonary arteries and
veins, at any developmental time point. In contrast, we observed in the
esophagus a high level of transgene expression in the developing embryo
but no detectable expression in adult mice, despite persistence of
transgene expression in many other SMC tissues (eg, airways, intestine,
coronary arteries, and small arterioles and veins). Finally, we
observed heterogeneity in expression between adjacent
individual SMCs within a given SM tissue and between blood vessels that
lie in proximity. These apparent differences in transgene expression
may simply reflect limitations of the methodology of detection; ie,
heterogeneity may be a function of the sensitivity of
the ß-galactosidase assay rather than reflecting distinct SMC
subpopulations that do or do not express the transgene. Importantly,
heterogeneity of expression of
SM-MHC45 and SM -actin46
within aortic SMCs of newborn animals has been reported on the basis of
immunohistochemical studies, suggesting that there may also be
differences in expression of these endogenous contractile
protein genes at least during early postnatal development. However,
heterogeneity of lacZ transgene
expression was observed in adult SM tissues in which 100% of the SMCs
showed detectable SM-MHC antibody staining (eg, the aorta and jejunum;
Figure 4). Clearly, the ability to detect SM-MHC gene expression is
highly dependent on (1) whether one attempts to detect expression at
the transcriptional or the translational level and (2) the sensitivity
of the detection method used. Indeed, such differences in detection
methodology may explain the apparent discrepancies between the
developmental time course of expression of the SM-MHC transgene in the
present study compared with detection of SM-MHC transcripts
reported by Miano et al.23 For example, in the
present study we first detected expression of the SM-MHC transgene
within the airway SMCs at E12.5 and, subsequently, in the esophageal,
aortic, and gastrointestinal SMCs at E14.5. In contrast, SM-MHC
transcripts were first detected by in situ hybridization in the aorta
at E10.5 and only later in airway and visceral
SMCs.23 One must therefore be very cautious in
interpreting results that involve use of very different methods with
poorly defined sensitivities. Regardless of this and its potential
implications, it remains to be reconciled why some SMCs in the
4.2+intron-lacZ mice were so strongly positive for
transgene expression and others were tremendously less so, even within
the same tissue.
There are a number of potential mechanisms, which are not mutually exclusive, that might explain the observed heterogeneity in SM-MHC transgene expression. First, it is possible that differences in transgene expression are the result of there being a requirement for distinct transcriptional regulatory cassettes or programs between SMC subtypes or lineages. This is analogous to a suggestion by Li et al,17 who found that expression of a transgene consisting of the initial 445 bp of the 5'-flanking region of the SM22 promoter coupled to lacZ was highly restricted to arterial SMCs in the adult mouse. In contrast, no expression was observed in small arteries and arterioles or in venous or visceral SMCs. At present, there are no known SMC lineages that correlate with this restricted pattern of SM22 transgene expression. However, of interest, we observed the virtual absence of expression of the 4.2+intron-lacZ transgene in pulmonary vessels, portions of the outflow tracts of the heart, and many of the major vessels in the head and neck, all of which are believed to include a neural crest component.47 48 As such, it is possible that at least part of the regional heterogeneity of transgene expression observed reflects lineage-dependent differences in transcriptional control programs and that additional genomic regions are required for expression in different subsets of SMCs. A second possibility is that regional differences in SM-MHC transgene expression may reflect differences in the milieu of local environmental factors that influence SM-MHC gene expression. However, it is difficult to reconcile the "microheterogeneity" we observed between immediately adjacent SMCs within a given blood vessel or SM tissue or between microvessels that lie in proximity, either on the basis of differences in local environmental factors or on the basis of different SMC lineages, at least any that have been described to date. A third possibility that must be considered is that at least some of the microheterogeneity observed may be the result of SM-MHC gene expression being episodic; ie, expression of SM-MHC within a given SMC may not be continuous but may occur in episodic bursts that are asynchronous across different cells in a given SM tissue. An intermittent pattern of transcription may be particularly appropriate for genes, such as SM-MHC, that encode for proteins that are believed to turn over relatively slowly in vivo and that are expressed at relatively constant levels in mature SMCs. Although at present there is no direct evidence that feedback regulation of SM-MHC expression occurs in SMCs, such controls must exist given the very precise regulation of SM-MHC concentrations within the SMC. Whereas it is well established that posttranscriptional controls exist for controlling intracellular levels of cytoskeletal proteins, such as tubulin,49 evidence of intracellular feedback controls of transcription of major cytoskeletal and contractile proteins is currently lacking. As a fourth and final explanation for the mosaic pattern of expression, it is possible that the lack of uniform expression is a penetrance problem related to the integration locus (ie, insertional variegation) as has been postulated for a number of other transgenes expressed in mice.50 This seems unlikely, however, since (1) all four independent transgene lines we studied showed a virtually identical expression pattern, and (2) this would require some inherent difference between SMCs within a given tissue that could account for variable penetrance for a given site of transgene insertion.
The finding that the lacZ transgene was highly expressed in the esophagus during embryogenesis and was later undetectable in the adult may be the result of the rare phenomenon known as "transdifferentiation." Using multiple skeletal and SM-specific markers (including SM-MHC), Patapoutian et al51 demonstrated that esophageal muscle tissue changes (ie, "transdifferentiates") from an SM phenotype to a skeletal muscle phenotype during the late fetal to early postnatal stage in development. The fact that this transition in phenotype was closely mimicked by the esophageal expression pattern of the SM-MHC transgene supports the transdifferentiation data and further suggests that the p4.2+intron-lacZ construct contained sufficient sequence for proper regulation in this tissue type. This would include any repressor elements that might be used for downregulation of transcription. Further analysis of additional developmental time points between E19.5 and the first 2 weeks after birth should allow us to determine whether transgene expression decreases in a rostrocaudal progression that was described for the transdifferentiation process. The testing of additional deleted or mutated SM-MHC-lacZ constructs may also provide further insight into the molecular regulation of this process.
The SM-MHC data in the present study add to the very recent, yet
rapidly growing, list of SM-specific/selective genes whose regulatory
programs are being investigated in the transgenic mouse. These genes
include SM -actin,52
SM22,18 53 telokin,21
and now SM-MHC. It will be of future interest to test whether sequence
elements found to be common among these genes are functionally
equivalent, especially with regard to similar and different SMC tissue
types. Of particular interest is the CC(A/T)6GG
element, or CArG-box, which binds to serum response factor and is
highly conserved and present in multiple copies in each of these
genes. To date, only for the SM22 gene has this element been
specifically mutated and shown to function as a required element in
vivo.53 The identification in the present
study of sufficient regions of the SM-MHC gene to drive SMC-specific
expression in transgenic mice provides for the first time the
appropriate context with which to begin to investigate the importance
of the SM-MHC CArG elements as well as a variety of other
cis elements shown to be important in regulation of this
gene in cultured SMCs.11 12 13 14 15 16 In addition, of
practical significance, the SM-MHC promoter-intronic fragment that we
have characterized herein represents the first genomic
construct that exhibits complete SMC-restricted expression in vivo. As
such, it may provide the basis for design of SMC-specific gene
targeting vectors for use in experimental animal models and for gene
therapy in humans.
|
Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
This study was supported by grants R01 HL-38854 and P01 HL-19242 from the National Institutes of Health (Dr Owens), by training grant 5T32-HL-07284 (Drs Madsen and Regan), by a fellowship grant from the Virginia Affiliate of the American Heart Association (VA-95-F18, Dr Madsen), by funds from the University of Virginia Cardiovascular Research Center, and by an Academic Enhancement Program Grant entitled "Gene Transfer and Gene Therapy in the Cardiovascular System" from the Office of the Associate Provost for Research at the University of Virginia. We gratefully acknowledge the expert technical assistance of Diane Raines, Andrea Tanner, and Margaret Ober.
Received January 23, 1998; accepted March 6, 1998.
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References |
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C. P. Mack and G. K. Owens Regulation of Smooth Muscle {alpha}-Actin Expression In Vivo Is Dependent on CArG Elements Within the 5' and First Intron Promoter Regions Circ. Res., April 16, 1999; 84(7): 852 - 861. [Abstract] [Full Text] [PDF]
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M. Aikawa, E. Rabkin, S. J. Voglic, H. Shing, R. Nagai, F. J. Schoen, and P. Libby Lipid Lowering Promotes Accumulation of Mature Smooth Muscle Cells Expressing Smooth Muscle Myosin Heavy Chain Isoforms in Rabbit Atheroma Circ. Res., November 16, 1998; 83(10): 1015 - 1026. [Abstract] [Full Text] [PDF]
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B. Camoretti-Mercado, H.-W. Liu, A. J. Halayko, S. M. Forsythe, J. W. Kyle, B. Li, Y. Fu, J. McConville, P. Kogut, J. E. Vieira, et al. Physiological Control of Smooth Muscle-specific Gene Expression through Regulated Nuclear Translocation of Serum Response Factor J. Biol. Chem., September 22, 2000; 275(39): 30387 - 30393. [Abstract] [Full Text] [PDF]
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A. M. Hoggatt, A. M. Kriegel, A. F. Smith, and B. P. Herring Hepatocyte Nuclear Factor-3 Homologue 1 (HFH-1) Represses Transcription of Smooth Muscle-specific Genes J. Biol. Chem., September 29, 2000; 275(40): 31162 - 31170. [Abstract] [Full Text] [PDF]
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P. J. Adam, C. P. Regan, M. B. Hautmann, and G. K. Owens Positive- and Negative-acting Kruppel-like Transcription Factors Bind a Transforming Growth Factor beta Control Element Required for Expression of the Smooth Muscle Cell Differentiation Marker SM22alpha in Vivo J. Biol. Chem., November 22, 2000; 275(48): 37798 - 37806. [Abstract] [Full Text] [PDF]
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M. Strobeck, S. Kim, J. C. L. Zhang, C. Clendenin, K. L. Du, and M. S. Parmacek Binding of Serum Response Factor to CArG Box Sequences Is Necessary but Not Sufficient to Restrict Gene Expression to Arterial Smooth Muscle Cells J. Biol. Chem., May 4, 2001; 276(19): 16418 - 16424. [Abstract] [Full Text] [PDF]
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J. M. Miano, C. M. Kitchen, J. Chen, K. M. Maltby, L. A. Kelly, H. Weiler, R. Krahe, L. K. Ashworth, and E. Garcia Expression of human smooth muscle calponin in transgenic mice revealed with a bacterial artificial chromosome Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1793 - H1803. [Abstract] [Full Text] [PDF]
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P. Qiu and L. Li Histone Acetylation and Recruitment of Serum Responsive Factor and CREB-Binding Protein Onto SM22 Promoter During SM22 Gene Expression Circ. Res., May 3, 2002; 90(8): 858 - 865. [Abstract] [Full Text] [PDF]
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