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(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 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) ( 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 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
A bovine B-myb cDNA plasmid expression vector, pB14,
was isolated from a custom cDNA library, constructed by Stratagene (La
Jolla, CA), in 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 pSV
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
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),
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
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
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
Figure 7:
Effects of B-myb expression on
the activity of the
The expression of the
Figure 8:
Effects of bovine B-myb expression on the activity of the
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
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 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 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 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.
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3359-3365
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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.-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). 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.
Cell Culture and Treatment Conditions
Smooth
muscle cell explants were derived from the aortas of female calves, as
we have described previously (Beldekas et al., 1982). Cultures
were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 1% nonessential amino
acids, 1% sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml
streptomycin (Life Technologies, Inc.). The medium was changed every
2-3 days, and cells were not used beyond the fourth passage. SMC
cultures were synchronized as described previously (Kindy and
Sonenshein, 1986). Briefly, cells were plated at low density (5 
10
cells/150-mm
dish) and allowed to grow
exponentially for 3 days, at which time the medium was changed to DMEM
supplemented with 0.5% FBS. Cells were maintained in 0.5% FBS for 3
days to achieve quiescence, at which time the cells were then
stimulated with fresh DMEM containing 10% FBS. With this procedure
(Kindy and Sonenshein, 1986), we have found that only 1-2% of
SMCs deprived of serum for 72 h demonstrate significant
[
H]thymidine nuclear labeling. Serum stimulation
results in an increase in labeled nuclei at 12 h, indicating the
beginning of DNA synthesis, with percent nuclear labeling increasing to
95% after 20 h of serum stimulation (Kindy and Sonenshein, 1986; Brown et al., 1992). Levels of histone H3.2 mRNA, an S
phase-specific gene, and cytofluorometric measurements further
confirmed cell synchrony (Kindy and Sonenshein, 1986). Alternatively,
serum-deprived cells were stimulated with either 20 ng/ml EGF or 100
nM 12-O-tetradecanoylphorbol-13-acetate (TPA) in the
absence or presence of 35 ng/ml insulin-like growth factor-1 (IGF-1)DNA Constructs
cDNA constructs employed were as
follows: 1(I) collagen, pCg54, a chicken cDNA clone (Lehrach et al., 1979); 1(V) collagen, pTV302, a human cDNA clone
(Takahara et al., 1991); B-myb, -B-myb,
a human cDNA clone (Nomura et al., 1988); histone H3.2,
pRAH3.2, a genomic fragment encoding amino acids 57-125 of
histone H3.2 (Alterman et al., 1984); and ornithine
decarboxylase, murine cDNA clone pOD48 (McConlogue et al.,
1984).
-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).
CAT 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).Transfections and Reporter Gene Assays
Cells were
plated at a density of 5 10 cells/100-mm dish 24 h before transfection. The medium was changed 2-4 h
before transfection. DNA (50 µg) was transfected by the modified
CaPO
transfection procedure of Chen and Okayama(1987).
Cells were harvested 48-72 h after transfection, and lysates were
prepared as described previously (Lawrence et al., 1994a).
Protein concentrations of the lysates were determined using the
Bradford assay as directed by the manufacturer (Bio-Rad). Equal amounts
of total protein were incubated with 2.5 µCi of
[H]acetyl coenzyme A (DuPont NEN; 200 mCi/mmol),
50 µM acetyl coenzyme A, and 1.6 mM chloramphenicol for 4-8 h, and the acetylated forms were
extracted with ethyl acetate and assayed by liquid scintillation
counting (Lawrence et al., 1994a).RNA Isolation and Hybridization Analysis
Total
cellular RNA was isolated according to the method of Chirgwin et
al. (1979) or with Tri-Reagent (Molecular Research Center, Inc.).
Equal quantities of RNA (15-25 µg) were denatured and
separated by electrophoresis on 1.0% agarose-formaldehyde gels.
Separated RNA was transferred onto a GeneScreen Plus nylon membrane
(DuPont NEN). RNA was cross-linked to the membrane by UV irradiation
(Stratalinker, Stratagene) at 0.12 J/cm for 30 s. For RNA
stability studies, cells were treated with 5 µg/ml actinomycin D
(Boehringer Mannheim). Probes were prepared as described previously by
Feinberg and Vogelstein(1982); hybridization reactions contained
1-2 10
cpm of P-labeled DNA/ml
of buffer. Unhybridized probe was removed by washing blots at 68 °C
with 2
SSC (1 SSC is 0.15 M NaCl, 15 mM sodium citrate), 0.1% SDS for 30 min, followed by 15-30 min
washes with 1 and 0.5 SSC, as needed. Quantitation by scanning
densitometry was performed using a Molecular Dynamics 300A computing
densitometer.Transcription Analysis
Nuclei were isolated from
SMCs, and run-off analysis was performed by a modification of the
method of Greenberg and Ziff(1984). Briefly, 1
10
nuclei were incubated in the presence of 250 µCi of
[P]UTP (DuPont NEN; 3200 Ci/mmol) for 30 min at
30 °C. Labeled RNA was isolated, and equal amounts of radiolabeled
RNA (4.5
10 cpm/ml of hybridization buffer) were
hybridized to plasmid DNA (10 µg/sample) immobilized onto
GeneScreen Plus by slot blotting followed by UV irradiation; after
hybridization, blots were washed as described above.
Expression of the B-myb mRNA in Aortic SMC
Cultures
Expression of B-myb in hematopoietic cells and
3T3 fibroblasts has been shown to be cell cycle-regulated, with
increasing levels detected during the G to S phase
transition (Golay et al., 1991; Lam et al., 1992). To
determine whether aortic SMCs express B-myb, Northern blot
analysis was performed using RNA isolated from bovine aortic SMCs
synchronized using the serum deprivation-stimulation protocol described
previously (Kindy and Sonenshein, 1986; see ``Materials and
Methods''). Serum-deprived quiescent cells begin to enter S phase
12 h after serum stimulation, and DNA synthesis peaks between 16
and 20 h (Kindy and Sonenshein, 1986). RNA was isolated from SMCs in
exponential growth, in quiescence, and at various times following serum
restimulation. Using a human B-myb cDNA clone as a probe,
Northern blot analysis revealed that exponentially growing SMCs express
significant quantities of an
3.5-kb B-myb mRNA (Fig. 1). In contrast, very low levels of B-myb RNA
were seen in quiescent SMCs, and RNA levels remained low until 8 h
after serum stimulation. Between 8 and 14 h of serum stimulation,
levels of B-myb RNA began to increase and continued to do so
until 24 h after serum stimulation. The level of B-myb mRNA
increased
6-8-fold during this time as judged by
densitometric scanning. Entry into S phase between 12 and 16 h was
verified using incorporation of [
H]thymidine and
appearance of histone H3.2 mRNA as described previously (Kindy and
Sonenshein, 1986) (data not shown). Therefore, B-myb is
expressed in bovine aortic SMCs in a cell cycle-dependent manner.
B-myb RNA levels are low in quiescence, increase in the mid to
late G phase of the cell cycle, immediately prior to the
onset of DNA synthesis, and continue to increase throughout S phase.
-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.
Regulation of B-myb Gene Transcription
To
determine whether transcriptional control mechanisms play a role in the
increase in steady-state B-myb RNA levels, nuclear run-off
analysis was performed. Nuclei were isolated from cells shortly after
serum stimulation (0.5 h), to allow adequate time for protein synthesis
to recover from the low levels in quiescent cells, and at 12 and 18 h
after serum stimulation, to compare transcription rates in late G and mid S phase. Hybridization to the B-myb probe
increased significantly between 0.5 and 12 h and remained constant
between 12 and 18 h (Fig. 2). In two separate experiments, an
1.6-2-fold increase was seen. In contrast, transcription of
the
1 chains of collagen types I and V decreased between 12 and 18
h and between 0.5 and 12 h, respectively. Transcription of ornithine
decarboxylase and histone H3.2 increased by 12 h, as expected
(McConlogue et al., 1984; Kindy and Sonenshein, 1986). These
results indicate that there is an increase in the rate of B-myb gene transcription during the cell cycle; this increase can
account for a part of the increase in steady-state RNA levels seen by
Northern analysis.
1(I) collagen, 1(V) collagen, and pUC19
plasmid DNA.
B-myb mRNA Decays Slowly
To assess the half-life
of B-myb mRNA, the rate of decay of B-myb RNA was
determined following addition of the transcriptional inhibitor
actinomycin D to SMCs in S phase. At 18 h after serum stimulation,
cells were treated for 1-4 h with 5 µg/ml actinomycin D,
which we have found effectively inhibits RNA synthesis (Kindy and
Sonenshein, 1986). No decay of B-myb RNA was observed even
after 4 h of actinomycin D treatment (Fig. 3). In contrast,
levels of the S phase-expressed histone H3.2 mRNA, which were
significant at 18 h, decayed substantially during this treatment,
consistent with results obtained previously (Brown et. al.,
1992). Therefore, B-myb mRNA appears relatively stable at the
time points where substantial steady-state levels are present.
Activation of B-myb by EGF and TPA plus IGF-1
We
next investigated the ability of specific growth factors to induce
expression of B-myb RNA. Subconfluent SMC cultures were made
quiescent via serum deprivation and were then stimulated with addition
of 20 ng/ml EGF. This growth factor is a weak mitogen for subconfluent
SMCs, as evidenced by the appearance of histone H3.2 mRNA, which was
detectable by 24 h (Fig. 4). EGF treatment resulted in a low
level of induction of B-myb mRNA that began to be detectable
at 12 h and was clearly seen at 24 h (Fig. 4). Thus, expression
of B-myb RNA was inducible by EGF and appeared to precede
entry into S phase, consistent with the G/S-phase specific
nature of the B-myb expression observed above.
-B-myb cDNA and histone H3.2 (His H3.2)
genomic clones, respectively; B, ethidium bromide-stained gel
confirming RNA quality and equal loading.
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.
Activity of B-myb as a Transcriptional
Regulator
Previously, we had shown that the reporter plasmid
KHK-CAT-dAX, derived by insertion of nine copies of the MBS directly in
front of the TK promoter linked to the CAT gene in the plasmid
dAX-TK-CAT, was transcriptionally active in SMCs (Brown et
al., 1992). To begin to assess the specific functional role of
B-myb in transactivational control in the vascular SMC, a
3.4-kb bovine pB14 B-myb cDNA clone, in the pBK-CMV plasmid
expression vector, was isolated from an aortic SMC cDNA library and
used in cotransfection experiments. SMC cultures, at 50%
confluence, were cotransfected with the KHK-CAT-dAX reporter construct
and increasing amounts of pB14 expression vector. After 3 days,
extracts were prepared, and equal amounts of proteins were analyzed.
B-myb expression resulted in dose-dependent down-regulation of
KHK-CAT-dAX activity, with a maximal decrease of
2.5-fold or 60% (Fig. 6). An average of three separate experiments yielded a
drop in activity of 60.3 ± 3.7%. In contrast, pB14 had no effect
on the activity of parental dAX-TK-CAT. Cotransfection with 12 µg
of pB14 only reduced dAX-TK-CAT activity to 92.0 ± 4.0% of
control in two separate experiments (data not shown). Cotransfection of
5 µg of a human pCEP-B-myb expression vector similarly
resulted in a specific down-regulation of the activity of KHK-CAT-dAX
(65%; data not shown). Thus, expression of B-Myb leads to repression of
the transcriptional activity of an MBS element-driven heterologous
promoter in SMCs.
dish).
Regulation of Collagen Promoters by
B-myb
Previously, we had noted that type I collagen mRNA levels
varied inversely with the growth state of the vascular SMC (Stepp et al., 1986; Kindy et al., 1988). Levels of type I
collagen mRNA were low in actively proliferating SMCs and increased in
quiescence due to either serum deprivation or growth to confluence.
Since bovine B-myb appeared to be a negative regulator of
transcription, we analyzed the promoters of the type I collagen genes
and identified several putative MBS elements in both the 1 and
2 chains of type I collagen (see ``Discussion''). Thus,
cotransfection experiments were performed to test the effects of
B-myb expression on collagen promoter activity. Increasing
concentrations of pB14 were cotransfected with the 2(I) collagen
promoter pMS-3.5/CAT construct, which contains 3.5 kb of sequence
upstream of the start site of transcription and 58 bp of exon 1 driving
the CAT reporter gene (Boast et al., 1990). The activity of
the pMS-3.5/CAT vector was down-regulated 3.8-fold (72%) upon
cotransfection with 10 µg of bovine B-myb vector (Fig. 7A). An average decrease of 82 ± 10.8% was
noted in three experiments upon cotransfection with 10 µg of pB14
DNA. Cotransfection with a human B-myb expression vector
similarly down-regulated the activity of the 2(I) promoter (Fig. 7B). The significance of the differences in the
slopes of the two curves is unclear given the differences in the
expression vectors used. An 4-fold (73%) down-regulation was
observed with 2.5 µg of human B-myb expression vector.
Thus, B-myb, either bovine or human, inhibits the activity of
the
2(I) collagen promoter in SMCs.
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.
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.
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.
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.
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. 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. () 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.
))
We thank Drs. Joe Lipsick, Francesco Ramirez, Barbara
Smith, David Rowe, Martino Introna, and T. Bender for generously
providing clone DNA reporter and expression constructs. The helpful
comments and suggestions of Dr. Marcello Arsura are gratefully
acknowledged. We also thank Dr. Judith Foster for use of the scanning
densitometer.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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