Published online before print July 5, 2001, doi: 10.1161/hh1401.093582
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© 2001 American Heart Association, Inc.
Article |
Prx1 Controls Vascular Smooth Muscle Cell Proliferation and Tenascin-C Expression and Is Upregulated With Prx2 in Pulmonary Vascular Disease
From the Department of Neurobiology (F.R.J., R.M.), Scripps Research Institute, La Jolla, and The Neurosciences Institute (D.B.E.), San Diego, Calif; Children’s Hospital of Philadelphia and Department of Pediatrics (R.J.O., P.L.J.), University of Pennsylvania School of Medicine, Philadelphia, Penn; and Department of Pediatrics (P.L.J.), Section of Critical Care, University of Colorado Health Sciences Center, Denver, Colo.
Correspondence to Peter Lloyd Jones, PhD, University of Colorado Health Sciences Center, 4200 East Ninth Ave, B-131, Denver, CO 80242. E-mail peter.jones{at}uchsc.edu
Abstract
Abstract—Prx1 and Prx2 are homeobox transcription factors expressed during vasculogenesis. To begin to elucidate how Prx1 and Prx2 are regulated and function in the adult vasculature, in situ hybridization studies were performed. Prx1 and Prx2 mRNAs were not detected in normal adult rat pulmonary arteries; however, both genes were induced with vascular disease, colocalizing to sites of tenascin-C (TN-C) expression. Because catabolism of the extracellular matrix (ECM) is a critical step in the development of vascular disease, we investigated whether changes in vascular smooth muscle cell (SMC)–ECM interactions regulate Prx1 and Prx2. A10 SMCs cultured on native type I collagen showed low levels of Prx1 and Prx2 mRNA expression, whereas cells cultured on denatured collagen showed higher levels of expression of both genes. At a functional level, transfection of SMCs with a Prx1 expression plasmid significantly increased their growth. Because TN-C also promotes SMC growth and its expression is also upregulated by denatured collagen, we tested and thereafter showed that Prx1 expression significantly enhances TN-C gene promoter activity 20-fold. Similar experiments conducted with truncated Prx1 proteins showed that the N-terminal portion and the homeodomain of Prx1 were necessary to induce the bulk of TN-C promoter activity. These findings support the hypothesis that Prx genes are regulated by changes in SMC adhesion and play key morphoregulatory roles during the development and progression of pulmonary vascular disease in adults.
Key Words: tenascin-C • homeobox genes • pulmonary
Homeobox transcription factors guide formation of the body plan during embryogenesis.1 Although homeobox genes also function during postnatal development and in adult disease,2 3 4 5 6 little is known about their roles in vascular remodeling.7 Prx1 and Prx2 represent paired-related homeobox genes,8 9 10 which characteristically bind class I homeodomain binding sites (HBSs) containing an ATTA core motif.11 Prx1 and Prx2 are expressed during embryogenesis, predominantly in mesenchyme-specific patterns.12 13 14 In the developing cardiovascular system, Prx1 and Prx2 are evident in the endocardial cushions and valves, the epicardium, and the wall of the great arteries and veins.14 15 In avian embryos, Prx1 and Prx2 are first expressed within the primary vessel wall of muscular coronary and pulmonary arteries (PAs), but as the vessels mature, their expression is restricted to nonmuscle cells in the adventitia and outer media.15
Among the targets that may be regulated by Prx proteins is the ECM protein tenascin-C (TN-C). In support of this, expression of Prx1, Prx2, and TN-C overlap in several settings including epithelial-mesenchymal transformation and vasculogenesis.16 TN-C is also expressed in remodeling adult tissues, including injured PAs, where it surrounds proliferating cells at the adventitial-medial boundary.17 18 Functionally, TN-C promotes growth and survival in cultured smooth muscle cells (SMCs) and in hypertensive PAs.19 20 On the basis of these findings, identifying the factors that control TN-C expression represents a potentially important step toward treating pulmonary vascular disease.
Multiple factors regulate TN-C, including ECM-degrading proteases.16 For example, inhibition of matrix metalloproteinase (MMP) activity suppresses TN-C expression and PA SMC growth, and reduces the severity of vascular lesions.19 20 These studies indicate that MMPs are upstream in an adhesion-dependent signaling pathway that controls TN-C. Consistent with this, we have shown that the TN-C gene promoter contains an ECM-responsive element that is silenced in SMCs cultivated on native type I collagen but is activated on the denatured form of this substrate.17 21 This ECM-responsive element harbors an HBS containing an ATTA core motif,22 which suggests that induction of TN-C expression by MMPs and denatured collagen might be controlled by homeobox proteins.
Here, we show that Prx1 and Prx2 are upregulated during the development of pulmonary vascular disease in adult rats, localizing to sites of TN-C expression. We also report that Prx gene expression is regulated by changes in SMC adhesion to type I collagen and that Prx1 controls SMC growth and TN-C gene transcription. These findings support the hypothesis that Prx proteins play key roles in the development of pulmonary vascular disease by controlling SMC proliferation and the composition of the vascular ECM.
Materials and Methods
Cell Culture
A10 vascular SMCs were maintained in M199. All
experiments were performed in triplicate, unless otherwise stated, in
M199 containing 2% FBS. Collagen substrates were prepared as
published.19 TN1-GFP
cells were generated by transfecting A10 SMCs with the TN1-pEGFP
construct. Cell lines were selected on the basis of resistance to G418
and via fluorescence-activated cell sorting for GFP. To
assess proliferation, SMCs were maintained in medium supplemented with
bromodeoxyuridine (BrdU) for the final 4 hours of the designated
experiment.
Prx Expression Vectors
Prx cDNAs were isolated by reverse transcribing
total RNA isolated from A10 SMCs. Truncated forms of rat Prx1 and Prx2
were also prepared using reverse transcriptase–polymerase chain
reaction (RT-PCR) and Pfu polymerase. cDNAs were cloned into a modified
pcDNA3 expression vector containing an N-terminal c-myc epitope
tag.
In Situ Hybridization
Adult Sprague-Dawley rat lung tissues were used for
in situ hybridization studies (a kind gift from Dr Marlene
Rabinovitch, The Hospital for Sick Children,
Toronto, Ontario, Canada). Riboprobes were generated using SP6
or T7 polymerase and [35S]dUTP. Sections
were incubated with riboprobes before signal detection with
photographic emulsion and were counterstained with Hoechst dye for
visualization of nuclei by epifluorescence. Prx mRNA expression
was visualized using dark-field microscopy.
mRNA Expression Studies
RT-PCR reactions were performed with cDNAs using
primer pairs for Prx1, Prx2, and GAPDH. For Northern analysis,
1 µg of A10 SMC Poly(A+) RNA was separated
and transferred to a nylon membrane. Hybridizations were performed with
32P–random-labeled Prx1 and Prx2 cDNA
probes. Autoradiograms were analyzed using
ImageQuant software. A loading control for RNA was also carried out by
comparing Prx mRNA expression with that of rat
GAPDH.
Western Immunoblotting
Protein was separated on 4% to 15%
polyacrylamide gels and transferred to polyvinylidene
difluoride membranes. The amount of protein loaded was
determined on the basis of transfection efficiency. To detect GFP and
c-myc, membranes were incubated with anti-GFP and anti-myc mouse
monoclonal antibodies. Membranes were then incubated with a horseradish
peroxidase–conjugated goat anti-mouse antibody and detected on Kodak
X-Omat film by enhanced chemiluminescence.
Immunostaining
For detection of Prx1, SMCs were incubated with an
anti–c-myc antibody, or with control IgG, and thereafter with an
FITC-conjugated species-specific antibody. Nuclei were detected with
DAPI. For BrdU detection, a cell proliferation assay kit was used.
BrdU-positive nuclei were scored in control- and Prx1-transfected SMCs,
and the percentage increases in BrdU incorporation determined after
accounting for differences in transfection
efficiency.
Site-Directed Mutagenesis
A 4173-bp fragment of the murine TN-C gene promoter
was ligated into pEGFP, a promoterless vector encoding GFP. This vector
was designated TN1-pEGFP. To assess whether the ATTA HBS is involved in
regulating TN-C promoter activity, site-directed mutagenesis was
performed using mutant oligonucleotide
primers.
Cotransfection and Luciferase Assays
The following plasmids were introduced using
LipofectAMINE: Prx1-pcDNA3 myc, TN1-pEGFP (wild-type), TN1-pEGFP
(mutant), pcDNAmyc empty vector, and pSV-ß–galactosidase
control reporter vector. Cells were harvested at 48 hours after
transfection and evaluated for transfection efficiency on the basis of
ß–galactosidase activity. Western immunoblotting was
then used for detection of GFP and myc proteins.
For luciferase reporter assays, A10 SMCs were transfected using Fugene reagent with either empty pcDNAmyc vector or the Prx1 expression vector, together with a TN-C promoter/luciferase reporter vector (TN7), containing the -247/+121 region of the murine TN-C gene containing the proximal promoter and a segment of the first exon.22 In these experiments, a lacZ reporter construct (CMVß) was cotransfected to provide an internal reference standard for transfection efficiency. Luciferase activity was measured using the Millipore Cytofluor 2450 system. To demonstrate that comparable levels of different Prx proteins are expressed after transfection of A10 cells, Western blots using the myc-tag antibody were performed on lysates of SMCs transfected with each Prx expression construct and the CMVß plasmid. In these experiments, the amount of lysate analyzed was first normalized to the ß-galactosidase activity.
Electromobility-Shift Assays
Wild-type and mutant oligonucleotides
encompassing the TN-C promoter HBS were end-labeled with
[
-32P]ATP. 0, 0.5, 1.5, and 4.5 µg of
nuclear protein were incubated with 20,000 cpm of
32-P labeled wild-type or mutant probe in
binding buffer. For supershift assays, Prx1-transfected A10 SMCs were
preincubated with anti-myc antibody before the addition of radiolabeled
wild-type or mutant oligonucleotide probes. Samples
were resolved on 7% nondenaturing acrylamide gels, and
DNA:protein complexes were visualized by
autoradiography.
Statistical Analyses
Results were compared by 1-way ANOVA and
Student-Newman-Keuls post hoc analysis. A
P value of <0.05 was
considered statistically significant.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
Results
Cloning of Rat Prx1 and Prx2
To isolate rat Prx genes, RT-PCR experiments were
performed using A10 SMC total RNA. A full-length Prx1 cDNA of 810 bp
was isolated containing an open reading frame of 651 bp, encoding a
217–amino acid protein. Comparison of this sequence with other Prx
gene sequences indicated that it was 100% identical to rat Prx1 (Rhox)
but also contained an additional exon of 72 bp that is found in several
mammalian Prx1 genes. This additional sequence is inserted at position
927 in the Rhox sequence. The Prx1 cDNA sequence is 97% identical to
that of mouse Prx1. The Prx2 cDNA from A10 cells was 570 bp and encodes
a protein that is 94% identical to mouse Prx2. This cDNA
represents a novel form of Prx2 that has a deletion of 57 amino
acids within the N-terminal region.
Induction of Prx1 and Prx2 With
Pulmonary Vascular Disease
To determine the expression patterns of Prx mRNAs,
riboprobes were hybridized to pulmonary tissue isolated from
adult rats injected with saline (control), or monocrotaline (MCT), an
alkaloid toxin that induces pulmonary
hypertension.17 Prx
antisense riboprobes did not hybridize to normal tissue
(Figure 1
), whereas these genes were expressed at the
adventitial–outer medial boundary in hypertensive animals by 21 days
after injection
(Figure 1). In addition, the airways and surrounding tissue
expressed Prx1 mRNA. By 28 days, Prx1 and Prx2 mRNAs were expressed in
the PA adventitia and within the media and
subendothelium
(Figure 1). Extensive Prx1 mRNA expression was also evident
in the airways and lung interstitium
(Figure 1). In contrast, Prx1 and Prx2 sense riboprobes did
not hybridize with either control or hypertensive tissue (data not
shown). Thus, Prx1 and Prx2 mRNAs are induced and upregulated with the
progression of pulmonary vascular
disease.
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Changes in SMC Adhesion Regulate Prx1 and
Prx2
Because remodeling of the PA ECM is critical to the
progression of vascular
disease,16 20 we
next sought to determine whether alterations in vascular SMC-ECM
interactions control Prx mRNA expression. As a model system, SMCs were
cultured on native and denatured type I collagen. A10 SMCs were used
for these studies because they behave in a manner that is identical to
that of primary PA SMCs in terms of their phenotypic and gene
expression responses to type I
collagen.19 21
RT-PCR studies showed that Prx1 and Prx2 mRNA expression was suppressed
by native collagen
(Figure 2A). In contrast, on denatured collagen, high levels
of both mRNAs were observed
(Figure 2A). Northern analyses for Prx1 and Prx2
mRNAs revealed Prx1 and Prx2 mRNA transcripts of 
4.5 and 1.3 kb,
respectively
(Figure 2B). Scanning densitometry of duplicate Northern
blots, normalized to GAPDH, showed that Prx1 and Prx2 were both
upregulated 3-fold in SMCs maintained on denatured collagen compared
with those maintained on native collagen
(Figure 2C). These results demonstrate that alterations in
SMC adhesion to the ECM control Prx1 and Prx2 mRNA
expression.
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Prx1 Promotes SMC Growth
To begin to establish a function for Prx1, SMCs
cultivated on denatured collagen were transiently transfected with a
Prx1 expression plasmid containing an N-terminal c-myc tag. Protein
expression was examined by Western blotting of A10 SMC nuclear extracts
and by indirect immunofluorescence using an
antibody against the c-myc tag. A Prx1 protein (26 kDa) was
expressed in nuclei of transfected cells
(Figure 3A). Immunofluorescence studies
showed that Prx1 was expressed in SMCs that appeared to be undergoing
division
(Figure 3B). Taken together with our observation that Prx1 is
upregulated in remodeling PAs, we hypothesized that Prx1 controls SMC
growth. To test this, A10 SMCs cultivated on native collagen were
cotransfected either with the parental c-myc vector or with the Prx1
vector and a ß-galactosidase–encoding expression plasmid in the
presence of BrdU to assess cell proliferation. When normalized for
transfection efficiency, overexpression of Prx1 significantly increased
BrdU incorporation by 78.1%
(P=0.03)
(Figure 3C).
|
Expression of TN-C Requires an HBS Within Its
Gene Promoter
Previously, we showed that TN-C is first expressed at
the adventitial-medial boundary of MCT-treated hypertensive rat PAs, as
well as in the airways and the
interstitium.17 This
expression pattern is identical to that observed for Prx1 and Prx2
mRNAs
(Figure 1
). Also consistent with our present
results obtained for Prx1 and Prx2 mRNA expression
(Figure 2), TN-C mRNA expression is reduced by native
collagen and is upregulated by denatured
collagen.19 21
Collectively, these data indicate that TN-C is upregulated with Prx1
and Prx2 in pulmonary vascular lesions and that changes in
vascular SMC adhesion alters the expression of all three
genes.
We have also shown that transcriptional induction of TN-C in
SMCs cultivated on denatured collagen requires a 122-bp DNA element in
the TN-C promoter containing an
HBS.21 To determine whether
the HBS is a key component in the ECM responsiveness of the TN-C
promoter, GFP-reporter constructs containing either the wild-type or an
HBS-mutated TN-C promoter were transiently transfected into SMCs
cultivated on native or denatured collagen. Mutation of the TN-C HBS
inhibited TN-C promoter activity in SMCs cultured on denatured
collagen, as compared with wild type-transfected SMCs
(Figure 4A). No GFP expression was observed in wild type- or
mutant- transfected SMCs cultivated on native collagen
(Figure 4A).
|
As a first step toward characterizing the protein(s) that
may bind to the HBS, electrophoretic mobility-shift assays were
performed. Radiolabeled oligonucleotides containing the
wild-type and mutated HBS were incubated with nuclear extracts prepared
from SMCs maintained on either native or denatured collagen. In SMCs
cultured on denatured collagen, a high molecular weight complex was
observed using the wild-type, but not the mutant, HBS
(Figure 4B). With SMCs cultured on native collagen, a low
molecular weight DNA:protein complex was observed when the wild-type,
but not when the mutant HBS was used as probe
(Figure 4B).
To demonstrate specificity of binding between the HBS and
nuclear extracts, reactions were carried out using either different
concentrations of nuclear extract or a 20- or 200-fold excess of
unlabeled competitor oligonucleotide. In these
experiments, the intensity of DNA:protein complexes observed with the
wild-type HBS increased with greater concentrations of nuclear extract;
formation of these complexes was abolished in reactions containing
excess unlabeled HBS
(Figure 4C). These experiments indicate that the TN-C
promoter HBS interacts with different proteins in an ECM-dependent
manner.
To determine whether Prx1 protein binds directly to the HBS, experiments were performed using radiolabeled HBS probes and nuclear extracts from cells transfected with the c-myc–tagged Prx1 expression vector. When compared with mock-transfected SMCs, no differences in DNA:protein complex formation were observed (data not shown). Preincubation of binding reactions with a c-myc antibody did not block or supershift DNA:protein complexes. Recombinant Prx1 protein also failed to bind to the wild-type HBS (data not shown). These results indicate that Prx1 either interacts weakly or does not bind directly to the core TN-C promoter HBS sequence.
Regions of the Prx1 Protein That Contribute
Toward TN-C Promoter Activation
Although Prx1 protein did not directly bind to
the probe containing the HBS, the coincident induction of Prx genes and
TN-C in pulmonary vascular disease and in isolated SMCs
prompted us to investigate whether Prx1 transactivates the TN-C
gene promoter. For these experiments, an A10 SMC line (TN1-GFP) was
generated in which a TN-C promoter-GFP reporter was stably integrated.
As shown in
Figures 5A and 5B, TN-C gene promoter activity was suppressed
on native collagen and activated on denatured collagen.
Transfection of TN1-GFP cells cultured on native collagen with the Prx1
expression plasmid led to increased Prx1 protein production and
TN-C promoter activity
(Figure 5C).
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To quantify the level of TN-C promoter activation by Prx1
and to determine the regions of Prx1 required for this activation,
cotransfection experiments were performed using a TN-C
promoter/luciferase gene reporter plasmid (construct
TN7)21 22 and
three different myc-tagged Prx1 expression constructs
(Figure 6A). These Prx constructs expressed either the
full-length Prx1 protein, a truncated form (PNL) containing the
N-terminal half of the protein with the Prx domain and nuclear
localization sequence, or a truncated form (PHD) containing the
N-terminal portion of Prx1 and the homeodomain.
|
To demonstrate that the constructs were expressed at
equivalent levels, Western blots were performed on extracts from A10
cells transfected with Prx1, PNL, and PHD constructs. Cellular
lysates were first adjusted to an internal reference standard of
ß-galactosidase activity. In a total of six experiments, no
differences in Prx protein expression levels were noted. A
representative Western blot is shown in
Figure 6B.
In luciferase reporter assays, Prx1 induced a 20-fold
induction of TN-C promoter activity relative to that observed in cells
transfected with empty pcDNA3-myc vector
(Figure 6C). The PHD construct led to a significant 10-fold
activation of the TN-C promoter, whereas the PNL construct produced a
<4-fold induction
(Figure 6B). These data indicate that the C-terminal portion
of Prx1 contributes 50% of the level of TN-C promoter activation.
However, the remaining segment of Prx1 containing the N-terminal
portion and homeodomain of Prx1 is required for high levels of TN-C
promoter activation. These data were collected from four separate
experiments performed in triplicate
(n=12).
Discussion
Although homeobox transcription factors control a range of cellular activities during embryonic development,1 2 3 4 5 6 7 8 little is known about their regulation and functions in adult tissues. We have shown that expression of two paired-related homeobox genes, Prx1 and Prx2, is induced during the development of pulmonary vascular disease in adult rats. This induction of Prx1 and Prx2 coincides with that of the ECM protein, TN-C. In addition, alterations in SMC adhesion were shown to regulate Prx gene expression. Because structural remodeling of the ECM also regulates SMC proliferation, TN-C biosynthesis, and the severity of pulmonary vascular disease,16 19 21 we assessed whether Prx1 could modulate these functions. Expression of Prx1 promoted SMC growth and induced TN-C expression. Finally, we showed that the ability of Prx1 to transactivate the TN-C promoter relies on distinct regions of this homeobox protein. These experiments support the idea that Prx proteins and concomitant alterations in the expression of particular ECM proteins (ie, TN-C) are likely to be important factors in the genesis of pulmonary vascular disease.
A tenable hypothesis based on this study is that expression of Prx and TN-C genes is controlled by the same factors. In keeping with this, bone morphogenetic proteins and angiotensin II23 24 25 have each been shown to regulate Prx and TN-C expression. In this study, we focused on the role of changes in SMC adhesion as a factor that controls Prx genes and TN-C. This direction was based on a growing body of evidence implicating cell adhesion as an important factor regulating vascular disease.16 Moreover, our previous work demonstrated that remodeling of native collagen activates a ß3 integrin–dependent extracellular signal–regulated kinase mitogen-activated protein kinase (ERK MAPK) signaling cascade that results in TN-C gene transcription.21 The present study indicates that Prx genes are also regulated by changes in the structure of type I collagen. It will therefore be important to determine whether ß3 integrin and ERK MAPKs also control the expression and/or posttranslational processing of Prx genes and proteins.
The expression of other homeobox proteins has also been shown to depend on the surrounding ECM. For example, endothelial cell HoxD3 expression is suppressed by basement membrane proteins during acquisition of an angiogenic phenotype.6 Similarly, HoxB7 expression in mammary epithelial cells is incompatible with basement membrane–directed lactational differentiation.2 Because HoxB7 is also expressed in fetal but not adult SMCs,4 it would be interesting to determine whether alterations in SMC HoxB7 expression are also influenced by changes in the vascular ECM.
Our studies show that Prx1 activates TN-C
transcription. This suggests that Prx proteins not only respond to
changes in cell adhesion, but they can also act in a reciprocal manner
to regulate the composition of the ECM. This type of "inside-out"
control has already been described for the Gax homeobox gene, which
promotes a quiescent SMC phenotype by suppressing expression of
vß3 and
vß5
integrins.5 Given that SMC
vß3 integrins also
interact with denatured
collagen19 (which we have
now shown promotes Prx1 and Prx2 gene expression), it is possible that
an inverse relationship exists between Gax and Prx genes in developing
and remodeling arteries.
The appearance of Prx1 and Prx2 in the adventitia of
hypertensive PAs suggests that these genes might regulate the behavior
of nonmuscular cells. Consistent with this, developmental
studies show that Prx gene expression and recruitment of SMCs first
takes place in the surrounding loose mesenchyme or primordial
adventitia.15 26
Whether further growth of the vessel wall involves proliferation of
medial SMCs or recruitment and growth of undifferentiated adventitial
cells is presently unknown. However, cell labeling studies clearly
show that adventitial fibroblasts represent a component of the
neointimal layer within injured adult systemic
arteries.27 In addition to
Prx1 and Prx2 expression, induction of –smooth muscle (SM) actin
represents another hallmark of activated fibroblasts in
different remodeling
tissues,28 and it has been
shown that Prx1 can transactivate the –SM actin gene
promoter.9 In light of the
present results, it will be important to determine whether Prx
genes also control adventitial fibroblast behavior in hypertensive PAs
via their ability to modulate –SM actin.
We also performed cellular transfection studies in SMCs cultivated on native collagen, a culture condition that suppresses endogenous Prx gene expression (present study) and proliferation.29 Overexpression of Prx1 produced significant increases in BrdU uptake. Although other studies indicate that specific Hox genes regulate cell growth during tumorigenesis,30 31 the present work is the first to show that paired-related homeobox genes control growth in a nontransformed adult tissue. In support of a growth-modulatory role for Prx1 protein, a yeast 2-hybrid screen with the N-terminal portion of p130, a member of the retinoblastoma (RB) gene family, identified Prx1 as an interacting transcription factor.32 Whether Prx1 inhibits or promotes RB function has not been determined. In vascular cells and tissues, however, overexpression of RB attenuates growth and neointimal formation.33 Perhaps interaction of Prx1 with RB favors cell cycle progression during vascular remodeling? Also in keeping with a growth-related role for Prx genes, selective expression of Prx2 in proliferating fibroblasts and the developing dermis correlates with TN-C expression and wound healing in fetal tissue.34 35
Our study also demonstrates that different Prx1 domains
contribute to TN-C gene transcription. One way that the N- and
C-terminal portions of Prx1 might participate in this transactivation
event is via interaction with a protein domain on another transcription
factor, or they might modulate the interaction of the Prx1 homeodomain
with other factors. Also, Prx1 activation of TN-C promoter activity
with lack of direct binding of Prx1 protein to the HBS suggests several
possibilities for transactivation. First, Prx1-HBS interactions might
require an accessory DNA element in the -247-bp TN-C promoter, which
binds a protein cofactor that is important to stabilize interaction of
Prx proteins with the HBS. Alternatively, Prx1 might not bind to the
HBS but instead interact with other proteins that are assembled at
other elements within the TN-C promoter. Such elements in the
-247/+121 TN-C promoter include binding sites for nuclear factor-1,
POU homeodomain proteins, nuclear factor-
B, and a TRE/AP-1 element
that binds to fos, jun, and other bZip
proteins.22 A tenable
hypothesis is that Prx1 activates TN-C transcription via
protein-protein interactions with these factors. For instance, Prx1 is
known to form complexes with the Maf oncoprotein, a bZip transcription
factor family member.36 A
Maf-recognition element, which is also a consensus TRE/AP-1 element, is
found at -114 bp in the TN-C promoter. Maf forms heterodimeric
combinations with Prx1, as well as with fos and jun; such interactions
may modulate the activity of TN-C and other genes that are targets of
these factors. Additionally, Prx1 may interact directly with components
of the basic transcription machinery assembled at the TATA box. For
instance, Prx1 is known to interact with the serum response factor and
RB,32 37 proteins
that are capable of interacting with the basic transcription machinery.
Alternatively, it is possible that Prx1 transactivates TN-C via
its ability to modulate the expression of transcription factors that
bind directly to the TN-C gene promoter.
Mice bearing null mutations in both Prx1 and Prx2 have been informative in elucidating the role of these genes during development.38 39 40 41 42 Mutant animals die 24 hours after birth and display skeletal and limb defects, as well as vascular anomalies, including abnormal positioning and awkward curvature of the aortic arch, and a misdirected and elongated ductus arteriosus. These defects are conceivably due to deregulated ECM synthesis.42 Whether this relates to altered TN-C expression, however, has not been determined.
In summary, our findings support the hypothesis that changes in SMC adhesion to the ECM are related to the expression and functions of Prx genes. In addition, the present work provides a foundation for studies aimed at deciphering the gene networks and signal-transduction events that are responsible for Prx gene expression through changes in the vascular ECM, as well as the mechanisms by which TN-C gene transcription and cellular proliferation are controlled under these conditions.
Acknowledgments
This work was supported by a grant from the National Science Foundation (to F.S.J.) and an American Heart Association Grant-in-Aid (to P.L.J.). We are grateful for the excellent technical assistance of Tom Moller.
Footnotes
Original received January 24, 2001; revision received May 22, 2001; accepted May 22, 2001.
References
1.
McGinnis
W. A century of homeosis, a decade of homeoboxes.
Genetics. 1994;137:607–611.
2.
Srebrow A,
Friedmann Y, Ravanpay A, Daniel CW, Bissell MJ. Expression of Hoxa-1
and Hoxb-7 is regulated by extracellular matrix-dependent signals in
mammary epithelial cells. J Cell
Biochem. 1998;69:377–391.
3.
Myers C, Charboneau
A, Boudreau N. Hox B3 promotes capillary morphogenesis and
angiogenesis. J Cell Biol. 2000;148:343–351.
4.
Miano JM, Firulli
AB, Olson EN, Hara P, Giachelli CM, Schwartz SM. Restricted expression
of homeobox genes distinguishes fetal from adult human smooth muscle
cells. Proc Natl Acad Sci
U S A. 1996;93:900–905.
5.
Witzenbichler B,
Kureishi Y, Luo Z, Le Roux A, Branellec D, Walsh K. Regulation of
smooth muscle cell migration and integrin expression by the Gax
transcription factor. J Clin
Invest. 1999;104:1469–1480.
6.
Boudreau N, Andrews
C, Srebrow A, Ravanpay A, Cheresh DA. Induction of the angiogenic
phenotype by Hox D3. J Cell
Biol. 1997;6:257–264.
7.
Gorski DH, Walsh K.
The role of homeobox genes in vascular remodeling and angiogenesis.
Circ Res. 2000;87:865–872.
8.
Cserjesi P, Lilly
B, Bryson L, Wang Y, Sassoon DA, Olson EN. Mhox: a mesodermally
restricted homeodomain protein that binds an essential site in the
muscle creatine kinase enhancer.
Development. 1992;115:1087–1101.
9.
Hautmann MB,
Thompson MM, Swartz EA, Olson EN, Owens GK. Angiotensin
II-induced stimulation of smooth muscle
10.
Kern MJ, Witte
DP, Valerius MT, Aronow BJ, Potter SS. A novel murine homeobox gene
isolated by a tissue specific PCR cloning strategy.
Nucleic Acids Res. 1992;20:5189–5195.
11.
Treisman J,
Harris E, Desplan C. The paired box encodes a second DNA-binding domain
in the paired homeo domain protein. Genes
Dev. 1991;5:594–604.
12.
Opstelten DJ,
Vogels R, Robert B, Kalkhoven E, Zwartkruis F, de Laaf L, Destree OH,
Deschamps J, Lawson KA, Meijlink F. The mouse homeobox gene, S8, is
expressed during embryogenesis predominantly in mesenchyme.
Mech Dev. 1991;34:29–41.
13.
Kuratani S,
Martin JF, Wawersik S, Lilly B, Eichele G, Olson EN. The expression
pattern of the chick homeobox gene gMHox suggests a role in patterning
of the limbs and face and in compartmentalization of somites.
Dev Biol. 1994;161:357–369.
14.
Leussink B,
Brouwer A, el Khattabi M, Poelmann RE, Gittenberger-de Groot AC,
Meijlink F. Expression patterns of the paired-related homeobox genes
MHox/Prx1 and S8/Prx2 suggest roles in development of the heart and the
forebrain. Mech Dev. 1995;52:51–64.
15.
Bergwerff M,
Gittenberger-de Groot AC, DeRuiter MC, van Iperen L, Meijlink F,
Poelmann RE. Patterns of paired-related homeobox genes PRX1 and PRX2
suggest involvement in matrix modulation in the developing chick
vascular system. Dev Dyn. 1998;213:59–70.
16.
Jones FS, Jones
PL. The tenascin family of ECM glycoproteins: structure,
function and regulation during embryonic development and tissue
remodeling. Dev Dyn. 2000;218:235–259.
17.
Jones PL,
Rabinovitch M. Tenascin-C is induced with progressive
pulmonary vascular disease in rats and is functionally related
to increased smooth muscle cell proliferation.
Circ Res. 1996;79:1131–1141.
18.
Jones PL, Cowan
K, Rabinovitch M. Tenascin-C, proliferation and
subendothelial accumulation of fibronectin in
progressive pulmonary vascular disease.
Am J Pathol. 1997;150:1349–1360.
19.
Jones PL, Crack
J, Rabinovitch M. Regulation of tenascin-C, a vascular
smooth muscle cell survival factor that interacts with the
ß3 integrin receptor to promote EGF receptor
phosphorylation and growth.
J Cell Biol. 1997;139:279–293.
20.
Cowan K, Jones
PL, Rabinovitch M. Elastase and matrix
metalloproteinase inhibitors induce regression and
tenascin-C antisense prevents progressive vascular disease.
J Clin Invest. 2000;105:21–34.
21.
Jones PL, Jones
F, Zhou B, Rabinovitch M. Denatured type I collagen
induction of vascular smooth muscle cell tenascin-C gene expression is
dependent upon a ß3 integrin-mediated
mitogen-activated protein kinase pathway and a 122 base pair
promoter element. J Cell
Sci. 1999;112:435–444.
22.
Copertino DW,
Edelman GM, Jones FS. Multiple promoter elements differentially
regulate the expression of the mouse tenascin gene.
Proc Natl Acad Sci
U S A. 1997;94:1846–1851.
23.
Stott NS, Jiang
TX, Chuong CM. Successive formative stages of precartilaginous
mesenchymal condensations in vitro: modulation of cell adhesion by
Wnt-7A and BMP-2. J Cell
Physiol. 1999;180:314–324.
24.
Hu YS, Zhou H,
Kartsogiannis V, Eisman JA, Martin TJ, Ng KW. Expression of rat
homeobox gene, rHOX, in developing and adult tissues in mice and
regulation of its mRNA expression in osteoblasts by bone morphogenetic
protein 2 and parathyroid hormone-related protein.
Mol Endocrinol. 1998;12:1721–1732.
25.
Hahn AW, Kern F,
Jonas U, John M, Buhler FR, Resink TJ. Functional aspects of vascular
tenascin-C expression. J Vasc
Res. 1995;32:162–174.
26.
Thayer JM, Meyers
K, Giachelli CM, Schwartz SM. Formation of the arterial
media during vascular development. Cell
Mol Biol Res. 1995;41:251–262.
27.
Li G, Chen SJ,
Oparil S, Chen YF, Thompson JA. Direct in vivo evidence demonstrating
neointimal migration of adventitial fibroblasts after
balloon injury of rat carotid arteries.
Circulation. 2000;101:1362–1375.
28.
Serini G,
Gabbiani G. Mechanisms of myofibroblast activity and phenotypic
modulation. Exp Cell Res. 1999;1:250:273–283.
29.
Koyama H, Raines
EW, Bornfeldt KE, Roberts JM, Ross R. Fibrillar collagen inhibits
arterial smooth muscle proliferation through regulation of
Cdk2 inhibitors.
Cell. 1996;87:1069–1078.
30.
Perkins A,
Kongsuwan K, Visvader J, Adams JM, Cory S. Homeobox gene expression
plus autocrine growth factor production elicits myeloid
leukemia. Proc Natl Acad Sci
U S A. 1990;87:8398–8402.
31.
Care A, Silvani
A, Meccia E, Mattia G, Stoppacciaro A, Parmiani G, Peschle C, Colombo
MP. HOXB7 constitutively activates basic fibroblast growth
factor in melanomas. Mol Cell
Biol. 1996;16:4842–4851.
32.
Wiggan O,
Taniguchi-Sidle A, Hamel PA. Interaction of the pRB-family proteins
with factors containing paired-like homeodomains.
Oncogene. 1998;16:227–236.
33.
Chang MW, Barr E,
Seltzer J, Jiang YQ, Nabel GJ, Nabel EG, Parmacek MS, Leiden JM.
Cytostatic gene therapy for vascular proliferative disorders with a
constitutively active form of the retinoblastoma gene product.
Science. 1995;27:267:518–522.
34.
Stelnicki EJ,
Arbeit J, Cass DL, Saner C, Harrison M, Largman C. Modulation of the
human homeobox genes PRX-2 and HOXB13 in scarless fetal wounds.
J Invest Dermatol. 1998;111:57–63.
35.
Whitby DJ,
Longaker MT, Harrison MR, Adzick NS, Ferguson MW. Rapid
epithelialisation of fetal wounds is associated with the early
deposition of tenascin. J Cell
Sci. 1991;99(pt 3):583–586.
36.
Kataoka K,
Yoshitomo-Nakagawa K, Shioda S, Nishizawa M. A set of Hox proteins
interact with the Maf oncoprotein to inhibit its DNA binding,
transactivation, and transforming activities.
J Biol Chem. 2001;276:819–826.
37.
Grueneberg DA,
Henry RW, Brauer A, Novina CD, Cheriyath V, Roy AL, Gilman M. A
multifunctional DNA-binding protein that promotes the formation of
serum response factor/homeodomain complexes: identity to TFII-I.
Genes Dev. 1997;11:2482–2493.
38.
Lu MF, Cheng HT,
Lacy AR, Kern MJ, Argao EA, Potter SS, Olson EN, Martin JF.
Paired-related homeobox genes cooperate in handplate and hindlimb
zeugopod morphogenesis. Dev
Biol. 1999;205:145–157.
39.
Martin JF,
Bradley A, Olson EN. The paired-like homeo box gene MHox is required
for early events of skeletogenesis in multiple lineages.
Genes Dev. 1995;15:1237–1249.
40.
ten Berge D,
Brouwer A, Korving J, Martin JF, Meijlink F. Prx1 and Prx2 in
skeletogenesis: roles in the craniofacial region, inner ear and limbs.
Development. 1998;125:3831–3842.
41.
Lu MF, Cheng HT,
Kern MJ, Potter SS, Tran B, Diekwisch TG, Martin JF. Prx-1 functions
cooperatively with another paired-related homeobox gene, prx-2, to
maintain cell fates within the craniofacial mesenchyme.
Dev Biol. 1999;205:145–157.
42.
Bergwerff M,
Gittenberger-de Groot AC, Wisse LJ, DeRuiter MC, Wessels A, Martin JF,
Olson EN, Kern MJ. Loss of function of the Prx1 and Prx2 homeobox genes
alters architecture of the great elastic arteries and ductus
arteriosus. Virchows Arch. 2000;436:12–19.
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