(Received for publication, August 14, 1995; and in revised form, November 3, 1995)
From the
The members of the Myb family of transcription factors are
defined by homology in the DNA-binding domain; all bind the Myb-binding
site (MBS) sequence (YG(A/G)C(A/C/G)GTT(G/A)). Here we report that
cultured bovine vascular smooth muscle cells (SMCs) express
B-myb. Levels of B-myb RNA found in exponential
growth were reduced dramatically in serum-deprived quiescent SMCs;
B-myb mRNA levels increased in the cell cycle during the late
G to S phase transition following restimulation with serum,
epidermal growth factor, or phorbol ester plus insulin-like growth
factor-1. Changes in the rate of B-myb gene transcription
could account for part of the observed increase following serum
addition. Treatment of SMC cultures with actinomycin D indicated a
>4-h half-life for B-myb mRNA during the S phase of the
cell cycle. Cotransfection of either a bovine or human B-myb expression vector down-regulated the activity of a multimerized
MBS element-driven reporter construct in SMCs. Putative MBS elements
were detected upstream of the promoters of the two chains of type I
collagen, which we have found to be expressed inversely with growth
state of the SMC (Kindy, M. S., Chang, C.-J., and Sonenshein, G.
E.(1988) J. Biol. Chem. 263, 11426-11430). In
cotransfection experiments, B-myb expression down-regulated
the promoter activity of
1(I) and
2(I) collagen constructs an
average of 92 and 82%, respectively. Thus, B-myb represents a
potential link in the observed inverse relationship between collagen
gene expression and growth of vascular SMCs.
The myb oncogene was first identified as the
transforming gene of two retroviruses, avian myeloblastosis virus and
E26, both of which cause myeloblastic leukemia in birds (Moscovici,
1975). In nontransformed cells, high levels of c-myb mRNA are
observed only in immature hematopoietic cells (Gonda and Metcalf,
1984), while lower myb levels have been detected in embryonic
neural tissue as well as neuroblastoma cells and in chick embryo
fibroblasts (Thiele et al., 1988; Thompson et al.,
1986). We found that cultured bovine vascular smooth muscle cells
(SMCs) ()also express low levels of c-myb mRNA
(Reilly et al., 1989; Brown et al., 1992). Two
c-myb related genes have been isolated based on their high
homology in the DNA-binding domains (Nomura et al., 1988; Lam et al., 1992). These genes, termed A- and B-myb, have
only recently begun to be characterized.
B-myb expression
has been detected in many tissues (Nomura et al., 1988; Golay et al., 1991; Arsura et al., 1992, 1994). Cell
synchronization studies have demonstrated that in 3T3 fibroblasts and
hematopoietic cells, B-myb displays a late
G-specific gene expression pattern, similar to that of
c-myb (Lam et al., 1992; Golay et al., 1991;
Reiss et al., 1991). Recent work has indicated that B-Myb
protein is capable of binding to the consensus Myb-binding site (MBS)
(YGRC(A/C/G)GTT(G/A)) (Howe and Watson, 1991), although R is preferably
T/C for c-Myb and A/C for B-Myb. Furthermore, B-Myb has also been
reported to recognize a second specific consensus sequence (CUNTTTCT)
as well (Mizuguchi et al., 1990). The transactivation
properties of B-myb are controversial, as one group reported
it to function as a positive regulator (Mizuguchi et al.,
1990), while several others had found it to be a transcriptional
inhibitor of c-myb-mediated transactivation (Foos et
al., 1992; Watson et al., 1993). These apparently
contradictory findings may be due to the fact that transfected
B-myb behaves differently in different cell lines (Tashiro et al., 1995). B-myb inhibited c-myb-induced
transactivation in 3T3 fibroblasts, whereas activation was observed
upon transfection into HeLa cells. Although no mechanism has been
established for this effect, Tashiro et al.(1995) proposed
that cell-specific expression of binding partner proteins allows for
differential formation of a functional dimer. In addition, B-myb has also been found to transactivate the DNA polymerase
gene
promoter independent of any identified MBS element (Venturelli et
al., 1990; Watson et al., 1993).
SMCs are the major cellular constituents of the medial layer of an artery, being responsible for maintaining vascular tone in the adult blood vessel (Ross, 1993). During the formation of a developing artery, SMCs display a synthetic phenotype; an initial highly proliferative phase is followed by synthesis of extracellular matrix components such as collagen, elastin, and proteoglycans (Hughes, 1942; Wu et al., 1992). This matrix provides a structural framework for the artery and also presumably allows for cell layering. Once the artery has been fully formed, SMCs differentiate into a contractile phenotype, in which they normally remain (Chamley-Campbell et al., 1979). As a normal response to injury and in certain disease states, however, SMCs migrate to the intimal layer, where modest rounds of proliferation are followed by production and deposition of matrix components over extended periods of time (Poole et al., 1971; Schwartz et al., 1985; Gordon et al., 1990; Ross, 1993; Strauss et al., 1994). These synthetic responses of SMCs, in association with deposition of lipids and minerals, can result in formation of an atherosclerotic plaque.
SMCs grown in culture
maintain a dedifferentiated synthetic phenotype. At low cell density,
they proliferate rapidly, but produce little connective tissue matrix
(Beldekas et al., 1982; Stepp et al., 1986). As we
and others have shown, production of connective tissue proteins, such
as collagen types I, III, and V, by SMCs increases dramatically as they
approach confluence, when their growth slows and cells begin to form
multilayers (Beldekas et al., 1982; Liau and Chan, 1989; Ang et al., 1990; Brown et al., 1991). Since vascular
SMCs express the c-myb oncogene, here we characterized
expression of B-myb. Aortic SMCs were found to express
B-myb in a cell cycle-dependent fashion; quiescent cells
contained low levels of B-myb RNA, with increasing levels seen
during the late G to S phase transition. Cotransfection of
B-myb expression vectors in SMC cultures inhibited the
activity of a multimerized MBS-driven heterologous promoter reporter
construct and of the promoters of the
1(I) and
2(I) collagen
genes, which contain putative elements for B-Myb binding. These
findings suggest that B-myb, which is expressed at high levels
in growing cells, may play a role in down-regulating collagen gene
expression in proliferating SMCs.
A bovine B-myb cDNA plasmid expression vector, pB14,
was isolated from a custom cDNA library, constructed by Stratagene (La
Jolla, CA), in -ZAP-EXPRESS using poly(A
) RNA
from exponentially growing aortic SMCs. The 3.4-kb insert of pB14 was
the largest isolated in the screening and represents a nearly
full-length cDNA based on the 3.5-kb B-myb mRNA size estimate.
Partial DNA sequence information of a PstI fragment of pB14
subcloned into the Bluescript vector, obtained by double-stranded
sequencing with Sequenase Version 2.0 (U. S. Biochemical Corp.),
indicated homology of >84% to the human B-myb sequences (bp
1755-1941) (Nomura et al., 1988), analyzed using the
Blast program (National Center for Biotechnology Information). The
human B-myb expression vector pCEP-B-myb contains the BamHI fragment, including the entire coding region of the
B-myb gene from plasmid pATB-18 (Arsura et al.,
1992), subcloned into the pCEP4
plasmid expression vector
(Invitrogen).
The reporter plasmid KHK-CAT-dAX was derived by
insertion of nine copies of the MBS directly in front of the thymidine
kinase promoter linked to the chloramphenicol acetyltransferase gene in
dAX-TK-CAT (Ibanez and Lipsick, 1990). The vector dAX-TK-CAT was in
turn constructed from pBLCAT2 by deletion of the AatII
polylinker (XhoI) fragment from the pUC18 plasmid backbone,
which appeared to confer a low level of myb-induced
transcription activity apparently caused by cryptic MBS elements
(Ibanez and Lipsick, 1990). The vector p1.6Bgl-CAT contains bp
-1114 to +513 of the murine c-myc gene linked to
the CAT reporter construct as described previously (Duyao et
al., 1992). pHNmyb-CAT contains 1 kb of sequence upstream of the
start site of transcription of the human c-myb promoter and
1.1 kb of exon 1 cloned into the pSVCAT vector (kindly
provided by T. Bender, University of Virginia School of Medicine,
Charlottesville, VA). The pMS-3.5/CAT construct contains bp -3500
to +58 of the human
2(I) promoter upstream of the CAT
reporter gene (Boast et al., 1990). The plasmid pOB3.6
contains 3.5 kb of the rat
1(I) collagen promoter plus the first
exon and first intron linked to the CAT reporter (Bedalov et
al., 1994). The plasmid ColCAT3.6 is composed of a 3.6-kb fragment
containing 3.5 kb of sequence upstream of the start site of
transcription and 115 bp of the first exon of the rat
1(I)
collagen gene linked to the CAT reporter (Lichtler et al.,
1989).
Figure 1:
Cell cycle expression of B-myb RNA in bovine vascular smooth muscle cells. Bovine aortic SMC
cultures were rendered quiescent by serum deprivation (DMEM plus 0.5%
FBS) for 72 h. Serum was then added back (DMEM plus 10% FBS) to allow
synchronous entry into S phase. Total RNA, isolated at the indicated
time points and from cells in exponential growth and quiescence, was
subjected to Northern blot analysis. A, autoradiogram of a
blot probed with the human -B-myb cDNA clone; B,
ethidium bromide-stained gel confirming RNA quality and equal loading. Lane E, exponential growth; lane Q, quiescence; lanes 2, 8, 14, 18, and 24, numbers indicate the hours after serum
addition.
Figure 2:
Nuclear run-off analysis of cell cycle
changes in the rate of transcription of the B-myb gene. SMC
cultures were made quiescent and serum-stimulated as described in the
legend to Fig. 1. Nuclei were isolated at 0.5, 12, and 18 h
after serum addition and subjected to nuclear run-off analysis. The
resulting radiolabeled RNAs were hybridized to the following cDNA
probes (10 µg) immobilized on nylon membranes: bovine B-myb (pB14), ornithine decarboxylase (ODC), histone H3.2 (His H3.2), 1(I) collagen,
1(V) collagen, and pUC19
plasmid DNA.
Figure 3: Decay of mRNA following actinomycin D treatment in the S phase of the cell cycle. SMC cultures were made quiescent and serum-stimulated as described in the legend to Fig. 1. At 18 h following serum addition, 5 µg/ml actinomycin D was added; total RNA was isolated after 1, 2, 3, and 4 h; and samples (15 µg) were subjected to Northern blot analysis for B-myb (pB14 cDNA clone) and histone H3.2 (His H3.2) mRNAs. RNA integrity and equal loading were confirmed by staining with ethidium bromide.
Figure 4:
Effects of EGF on expression of B-myb RNA in bovine vascular SMCs. Bovine aortic SMC cultures were
rendered quiescent by serum deprivation (DMEM plus 0.5% FBS) for 72 h.
EGF (20 ng/ml) was added, and total RNA was isolated at the indicated
time points and from cells in quiescence (Q) and subjected to
Northern blot analysis. A, autoradiogram of a blot probed with
human -B-myb cDNA and histone H3.2 (His H3.2)
genomic clones, respectively; B, ethidium bromide-stained gel
confirming RNA quality and equal loading.
Phorbol
ester treatment of quiescent cells has been found to induce genes
mediating competence, such as c-fos and c-myc, and
entry into the G phase of the cell cycle (Greenberg and
Ziff, 1984; Kelly et al., 1983). Further transit from G
into S phase requires stimulation with a progression factor, such
as IGF-1 (Leof et al., 1982). To examine the effects of these
agents on SMCs, serum-deprived quiescent cell cultures were stimulated
with 100 nM TPA in the absence or presence of 35 ng/ml IGF-1.
RNA was isolated from cells in quiescence (0 h) or 10, 16, and 24 h
after stimulation. B-myb RNA levels were low in quiescence (Fig. 5), as observed above (Fig. 1). No significant
increase in B-myb expression was seen with TPA treatment
alone. In contrast, B-myb RNA levels increased in the cells
treated with both TPA and IGF-1. Thus, treatment with TPA made SMCs
competent to respond to the progression factor IGF-1, leading to
increased expression of B-myb.
Figure 5: Effects of TPA in combination with IGF-1 on expression of B-myb RNA in bovine vascular SMCs. SMC cultures were rendered quiescent by serum deprivation (DMEM plus 0.5% FBS) for 72 h. Cells were treated with 100 nM TPA in the absence(-) or presence (+) of 35 ng/ml IGF-1. Total RNA was isolated at the indicated time points as well as from cells in quiescence (0 h) and subjected to Northern blot analysis. A, autoradiogram of a blot probed with the human B-myb cDNA clone probe; B, ethidium bromide-stained gel confirming RNA quality and equal loading.
Figure 6:
Activity of B-myb as a
transcriptional regulator of an MBS element-driven construct in SMCs.
Twenty-five micrograms of the reporter plasmid KHK-CAT, containing nine
MBS elements upstream of the TK promoter and CAT gene, were
cotransfected in duplicate into aortic SMCs with increasing amounts of
bovine B-myb expression vector pB14. pUC19 DNA was used to
equalize the total amounts of DNA transfected (50 µg/100-mm dish).
Figure 7:
Effects of B-myb expression on
the activity of the 2(I) collagen promoter in SMCs. SMCs were
transfected in duplicate with 25 µg of pMS-3.5/CAT collagen
2(I) promoter reporter construct (Boast et al., 1990) in
the presence of the indicated amounts of B-myb expression
vector and of pUC19 DNA to make up a total of 50 µg of
DNA/100-mm
dish. Extracts containing equal amounts of
protein were assayed for CAT activity. A, bovine
B-myb; B, human
B-myb.
The expression of the
1 and
2 genes of type I collagen is often coordinately
regulated (Stepp et al., 1986). Therefore, we assessed the
effects of B-Myb expression on the activity of the
1(I) promoter.
pOB3.6, which contains 3.6 kb of the
1(I) collagen promoter plus
all of exon 1 and intron 1 upstream of the CAT reporter gene, was
cotransfected with the bovine B-myb expression vector pB14.
The activity of pOB3.6 was down-regulated
8.8-fold, in a
dose-dependent manner, by coexpression of B-myb (Fig. 8). Cotransfection with 10 µg of bovine pB14
reduced pOB3.6 activity an average of 92 ± 2.5% in three
experiments, and that with 10 µg of human pCEP-B-myb expression vector reduced it 79% (data not shown). The plasmid
ColCAT3.6, which contains 3.5 kb of the
1(I) collagen promoter
plus 115 bp of exon 1 upstream of the CAT reporter gene, displayed
fairly low levels of CAT activity in SMCs (data not shown). This
activity was similarly down-regulated by the presence of either human
or bovine B-myb, but to a somewhat lesser extent, 57 ±
2.4% (data not shown). Therefore, B-Myb is a specific regulator of
transcription that is able to down-regulate the activity of the
promoters of the genes encoding both chains of type I collagen.
Figure 8:
Effects of bovine B-myb expression on the activity of the 1(I) collagen promoter in
SMCs. SMCs were transfected in duplicate with 25 µg of pOB3.6
collagen
1(I) promoter reporter construct (Bedalov et
al., 1994) in the presence of the indicated amounts of bovine
B-myb expression vector and of pUC19 DNA to make up a total of
50 µg of DNA/100 mm
dish. Extracts containing equal
amounts of protein were assayed for CAT
activity.
To
determine whether B-myb acts nonspecifically as a negative
regulator of transcription in SMCs, cotransfection analysis was
performed with the c-myc and c-myb promoters, both of
which contain MBSs that have been shown to be regulated by c-Myb (Evans et al., 1990; Nakagoshi et al., 1992; Nicolaides et al., 1991). Cotransfection of 5 µg of pB14 with the
c-myc promoter plasmid p1.6Bgl-CAT resulted in only an
15% reduction in its activity. These results agree with those of
Watson et al.(1993), who found that B-myb had no
effect on c-myc promoter activity in 3T3 fibroblasts.
Similarly, the activity of the c-myb promoter plasmid
pHNmyb-CAT was down-regulated only 12% upon cotransfection with
B-myb. Thus, B-myb expression did not appear to
significantly affect the promoter activity of these two oncogenes,
suggesting that the inhibition of collagen promoter activity described
above is specific.
Proliferating primary bovine aortic SMCs were found to
express B-myb, a member of the myb gene family.
B-myb expression in SMCs occurred in a cell cycle-dependent
fashion and displayed negative regulatory activity with respect to an
MBS element-driven construct and the 1(I) and
2(I) collagen
promoters. Quiescent SMCs expressed very little B-myb mRNA,
and levels increased as cells entered late G
and peaked in
S phase following stimulation with serum, EGF, or a combination of
treatment with TPA and IGF-1. Previous work in several laboratories,
including our own, demonstrated an inverse relationship between SMC
growth and collagen production (Jones et al., 1979; Stepp et al., 1986; Kindy et al., 1988; Liau and Chan,
1989; Ang et al., 1990; Chang and Sonenshein, 1991). When SMCs
were proliferating rapidly, collagen gene expression was low, whereas
when they were confluent or made quiescent via either serum starvation
or isoleucine deprivation, collagen mRNA expression increased
significantly. The findings presented here suggest the intriguing
possibility that B-Myb mediates signals regulating this inverse
relationship between growth and collagen gene expression in SMCs;
furthermore, they indicate that collagen genes represent a new family
of targets for regulation by a member of the myb gene family.
When transfected into aortic SMCs, bovine B-myb negatively regulated an MBS element/heterologous promoter-driven construct. Thus, the overexpression of B-myb can apparently override, presumably via competition for binding, the induction of MBS element activity by the low level of endogenously expressed c-myb previously noted in these cells (Brown et al., 1992). This is similar to results obtained in other cell types with the human and murine homologs of B-myb. In 3T3 cells, for example, murine B-myb has been shown to be a competitive inhibitor of c-myb-induced transactivation of an MBS-driven construct (Watson et al., 1993). GAL4 fusion studies have also shown that the C terminus of murine B-Myb has no intrinsic ability to transactivate when fused with the DNA-binding domain of the GAL4 protein. This lack of transactivating ability is not due simply to lack of DNA binding by B-Myb since gel shift and footprinting studies have shown that B-Myb was able to bind to an MBS. Recently, it has been shown that B-Myb is able to function as a strong transcriptional activator when transfected into certain cell types, such as HeLa cells (Tashiro et al., 1995). It has been postulated that this cell type specificity relates to the absence or presence of a cofactor that binds to B-Myb in its C-terminal conserved region and mediates transactivation.
The selective down-regulation of the activity of
the promoters for the two chains of type I collagen upon B-myb expression in transient cotransfection analysis in primary
cultured SMCs may occur by either a direct or an indirect mechanism.
DNA analysis of the rat and human collagen COL1A1 and A2 genes revealed
the presence of several putative Myb-binding sites and B-Myb-specific
sequence elements. The presence of these putative sites raises the
possibility of a direct effect of B-Myb on collagen genes. The limited
size of these sequences, however, necessitates more specific mapping
analysis. It should also be noted that indirect mechanisms have been
observed, e.g. with the DNA polymerase promoter
(Venturelli et al., 1990; Watson et al., 1993); these
could similarly be involved with the down-regulation of transcription
of collagen genes via expression of B-Myb and would be of equal
functional significance for matrix formation by the SMC. We have
recently found that co-microinjection of B-myb with c-myc expression vectors into quiescent SMCs failed to induce entry into
S phase, suggesting that the observed inhibition of collagen gene
transcription is not simply due to a change in the proliferative state
of the cell. (
)
The 6-8-fold increase in B-myb mRNA seen in the cell cycle can be partly accounted for by the
1.6-2-fold increase in the overall rate of transcription of the
gene. In 3T3 fibroblasts, the mechanism of the cell cycle increase in
B-myb RNA levels was determined to be due to an increase in
the rate of transcription of the gene. Deletion of an E2F site
abrogated cell cycle regulation of B-myb transcription (Lam
and Watson, 1993). Gel shift analysis revealed that quiescent cells
showed E2F binding that was supershifted only with antibodies to E2F
and p107, while in S phase, this complex contained cyclin A as well. It
is possible that other mechanisms play a significant role in B-myb expression in SMCs. Alternative levels of control include either a
change in the rate of elongation of RNA chains during synthesis or of
RNA processing or altered stability. For example, c-myb mRNA
levels in hematopoietic cells are controlled mainly by the rate of
elongation of transcription (Bender et al., 1987), while in
chick embryo fibroblasts, mRNA stability is the main level of
regulation (Thompson et al., 1986). Interestingly, the
increase in c-myb mRNA levels in SMCs during the late G to S phase transition could not be accounted for either by an
enhanced rate of gene transcription or by a change in the stability of
the c-myb RNA (Brown et al., 1992), suggesting
additional levels of control.
The observation that there is an inverse relationship between matrix deposition and cellular proliferation is a long standing one that has been substantiated in many different systems. For example, viral transformation of fibroblasts enhanced the proliferative capacity of these cells while decreasing their level of synthesis of type I collagen (Adams et al., 1982). Overexpression of the ras oncogene in Rat1 fibroblasts had a similar affect on type I collagen gene expression by these cells (Slack et al., 1992). In density-arrested nondividing human fetal lung fibroblasts, type I and III collagen mRNA levels were significantly higher than those in logarithmically growing cells (Miskulin et al., 1986). Thus, it appears that when genes necessary for growth, such as oncogenes, are expressed, other genes that are inconsistent with or unnecessary for growth are turned off. The fact that B-myb is expressed broadly in many different cell types presents the possibility that the signal transduction pathway that mediates activation of this gene may be involved in the inhibition of collagen gene expression in cells derived from many different tissues.
SMCs are responsible for synthesizing the extracellular matrix components in the medial layer of a normal artery, including collagen, elastin, fibronectin, and proteoglycans, as well as the enzymes involved in matrix protein deposition, such as lysyl oxidase (reviewed by Ross(1993)). During arterial development in the chick, an initial SMC synthetic phase is followed by deposition of matrix proteins and additional cell layering (Hughes, 1942; Wu et al., 1992). The most abundant collagen species produced by SMCs is type I collagen, with lesser but still significant amounts of collagen types III, V, and VI. In atherosclerosis, SMCs migrate from the medial layer to the intima, where some initial rounds of proliferation are followed by extensive synthesis and deposition of matrix proteins (Poole et al., 1971; Gordon et al., 1990; Ross, 1993; Strauss et al., 1994). The majority of the mass of a fibrous plaque is composed of the collagen proteins deposited by the SMC. The subsequent occlusion of the lumen of the artery and the clinical sequelae that follow are a primary cause of morbidity and mortality in the Western world. Coordinate regulation of many collagen species, including types I, III, and V, has been noted in SMC cultures under a variety of conditions that affect growth state (Jones et al., 1979; Stepp et al., 1986; Liau and Chan, 1989; Ang et al., 1990; Brown et al., 1991; Lawrence et al., 1994b). In addition, other genes necessary for matrix deposition, such as lysyl oxidase, have similar inverse expression patterns in relation to growth (Kenyon et al., 1991). Thus, the possibility that B-Myb plays a more general role in regulation of matrix gene expression in SMCs is under investigation.