Departments of 1 Physiology and Biophysics and of 2 Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120; and 3 Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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A cell-specific
promoter located in an intron of the smooth muscle myosin light chain
kinase gene directs transcription of telokin exclusively in smooth
muscle cells. Transgenic mice were generated in which a 310-bp rabbit
telokin promoter fragment, extending from 163 to +147, was used
to drive expression of simian virus 40 large T antigen. Smooth
muscle-specific expression of the T-antigen transgene paralleled that
of the endogenous telokin gene in all smooth muscle tissues except
uterus. The 310-bp promoter fragment resulted in very low levels of
transgene expression in uterus; in contrast, a transgene driven by a
2.4-kb fragment (
2250 to +147) resulted in high levels of
transgene expression in uterine smooth muscle. Telokin expression
levels correlate with the estrogen status of human myometrial tissues,
suggesting that deletion of an estrogen response element (ERE) may
account for the low levels of transgene expression driven by the 310-bp
rabbit telokin promoter in uterine smooth muscle. Experiments in A10
smooth muscle cells directly showed that reporter gene expression
driven by the 2.4-kb, but not 310-bp, promoter fragment could be
stimulated two- to threefold by estrogen. This stimulation was mediated
through an ERE located between
1447 and
1474. Addition of
the ERE to the 310-bp fragment restored estrogen responsiveness in A10
cells. These data demonstrate that in addition to a minimal 310-bp
proximal promoter at least one distal
cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal
element may include an ERE between
1447 and
1474.
myosin light chain kinase; estrogen response element; uterus
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INTRODUCTION |
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MANY PATHOLOGICAL conditions, such as atherosclerosis, restenosis following angioplasty, and chronic asthma, are associated with changes in the growth and differentiation state of smooth muscle (3, 18). These changes result in downregulation of many proteins characteristic of adult smooth muscle. Although the extracellular signals that influence the growth and differentiation state of smooth muscle have been studied extensively (27), little is known about the nuclear factors that control these processes. To begin to identify nuclear factors that regulate expression of smooth muscle contractile proteins, we initiated an analysis of mechanisms regulating the smooth muscle-specific expression of the rabbit telokin gene (8, 9).
Telokin is a 17-kDa acidic protein expressed exclusively in smooth
muscle tissues and cells (6, 8). Although the physiological function of
telokin is unclear, telokin has been shown to bind to nonphosphorylated
myosin filaments and to stimulate myosin mini-filament assembly in
vitro (10, 25). On the basis of these observations, it has been
proposed that, in vivo, telokin may bind to nonphosphorylated myosin
filaments and stabilize these filaments in resting smooth muscle (10,
25). The amino acid sequence of rabbit telokin is identical to the
carboxy-terminal 155 residues of rabbit smooth muscle myosin light
chain kinase (6, 8). The protein is not a proteolytic fragment derived from the smooth muscle myosin light chain kinase but is the translation product of a distinct 2.6-kb mRNA (8). Telokin mRNA is transcribed from
a second promoter, located within an intron, in the 3' region of
the smooth muscle myosin light chain kinase gene. A 2.4-kb fragment of
the rabbit telokin promoter has been shown to direct smooth
muscle-specific expression of a transgene, in vivo, in transgenic mice
(8). Expression of the transgene mirrored the expression of endogenous
telokin, demonstrating that all of the regulatory elements necessary to
mediate the cell-specific expression of telokin are located within the
2.4-kb promoter fragment. Deletion analysis of the promoter identified
a 310-bp fragment extending from 163 to +147 that is sufficient
to mediate maximal cell-specific reporter gene expression in cultured
cells (8). Several positively acting regulatory elements have been
identified within this region that are important for expression of the
promoter in A10 vascular smooth muscle cells (9). These include a TATA
box located between
69 and
64 and a single
CC(A/T)6GG (CArG) motif, which binds serum
response factor (SRF), located between
56 and
47, both of
which were found to be critical for high levels of telokin transcription.
In addition to the telokin promoter, the regulatory regions of SM22
(16) and smooth muscle
-actin (24) genes have also been shown to
direct transgene expression to smooth muscle tissues in vivo in
transgenic mice. These transgenes exhibit a distinct pattern of
expression in different smooth muscle tissues. Both the telokin and
-actin transgenes are expressed at high levels in visceral smooth
muscle and at lower levels in vascular smooth muscle. In contrast, the
mouse SM22
promoter [2.8-kb (
2735 to +63), 508-bp
(
445 to +63), or 321-bp (
280 to +41) fragments] directs transgene expression specifically to arterial smooth muscle in
adult mice. (13, 16). The patterns of expression of these transgenes in
various smooth muscle tissues suggest that distinct regulatory elements
may be required for expression of a single gene in different smooth
muscle tissues. These data also demonstrate that, although promoter
fragments may mediate cell-specific expression in vitro in cultured
vascular smooth muscle cells, additional regulatory elements may be
required in vivo in specific smooth muscle tissues.
In the current study, the activity of a 310-bp minimal rabbit telokin
promoter fragment, identified from assays in A10 smooth muscle cells,
was analyzed in vivo in transgenic mice. With the exception of uterine
smooth muscle, expression of the transgene mimics the endogenous
telokin gene. Transgene expression was barely detectable in uterine
smooth muscle of several founder animals. This result is in contrast to
a transgene driven by a 2.4-kb fragment of the telokin promoter, which
is expressed at high levels in uterine smooth muscle. Correlation
between telokin expression and estrogen levels in human myometrial
smooth muscle suggests that telokin expression in mammalian uterus may
be stimulated by estrogen. Using reporter gene assays in A10 smooth
muscle cells, we directly show that the rabbit telokin promoter
contains a nonconsensus estrogen response element (ERE) between
1474 and
1447. Deletion of this element may account for
the low levels of expression of the 310-bp transgene in uterine smooth
muscle. These data suggest that different smooth muscle tissues require
distinct cis-acting regulatory
elements to mediate expression of a single smooth muscle-specific gene.
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MATERIALS AND METHODS |
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Transgenic mice. The telokin promoter T310-simian virus 40 (SV40) large T-antigen transgene was generated by ligating a 310-bp Sac I-Kpn I fragment from T310-pGL2-Basic (8) into TAG-Bluescript (5). Transgenic mice were generated in C3HeB/FeQ inbred embryos by the Indiana University Transgenic Mouse Facility, using standard protocols.
Western and Northern blotting. Western and Northern blots for SV40 large T antigen and telokin were performed as described previously (6, 8).
Immunohistochemical staining.
Tissues were collected from transgenic mice and cryoprotected in 20%
sucrose-PBS overnight. Cryoprotected tissues were embedded in
tissue-freezing medium (TBS, Durham, NC) and frozen at 70°C; 8-µm cryosections were cut, fixed in 3.7% formaldehyde in PBS for 5 min, permeabilized in 0.2% Triton X-100 for 5 min, and then incubated
with the appropriate antibody for 2-6 h at 37°C. After extensive washing in 50 mM Tris (pH 7.6), 150 mM NaCl sections were
incubated with fluorescein conjugated to donkey anti-rabbit IgG for 1 h.
Plasmid constructions.
T2.4-pGL2-Basic (2250 to +147) and T310-pGL2-Basic (
163
to +147) telokin promoter luciferase vectors were described previously (8). A deletion of the ERE (
1492 to
1421) in the
T2.4-pGL2-Basic construct was achieved by oligonucleotide directed
loop-out mutagenesis (15). To generate the TERE-T310-pGL2-Basic
plasmid, a double-stranded oligonucleotide containing the ERE
(5'-ATTGACCTGCACAGGGTCACGCAACGGTCAGG-3') was ligated into
the Sac I site in the pGL2-Basic
polylinker 5' to the 310-bp telokin promoter fragment. ERE-TK (a
gift from E. Holler, Universität Regensburg, Regensburg, Germany)
contains a consensus ERE (GGTCACTGTGACC) located 5' to the
minimal thymidine kinase (TK) promoter (residues
109 to
+52) driving expression of the luciferase gene (20).
Cell transfections and reporter gene assays.
Luciferase fusion genes were transfected into rat A10 vascular smooth
muscle cells as described previously (8). For analysis of estrogen
effects on reporter gene activity, A10 cells were grown in phenol
red-free MEM (Bio Whittaker, Walkersville, MD) containing
10% charcoal-stripped fetal bovine serum, MEM nonessential amino acids
(1×; Sigma, St. Louis, MO), 2 mM glutamine, 10 mM HEPES, 50 U/ml
penicillin, and 50 µg/ml streptomycin. Cells to be transfected were
seeded at 2 × 105
cells/well in six-well plates. Twenty hours after seeding, each well
was washed twice with PBS and incubated with a cocktail containing 3 µg of plasmid DNA [1 µg promoter-luciferase plasmid, 1 µg
estrogen receptor expression plasmid (HEGO, a gift from P. Chambon,
Strasbourg, France; Ref. 14) or vector control, and 1 µg
cytomegalovirus (CMV)- galactosidase plasmid as an internal
control] and 5.5 µl of Lipofectamine in 1.5 ml of Optimem
(GIBCO BRL). After 8 h the transfection cocktail was replaced with
growth medium containing 17
-estradiol
(10
8 M) or vehicle. Cell
extracts (200 µl/well) were prepared for luciferase and
-galactosidase activity assays 24 h later.
Human myometrial samples. Normal human myometrial tissue was removed from the uterus of nonpregnant women after hysterectomy for benign gynecologic conditions. In cases of pregnancy, myometrium was obtained at the time of cesarean-hysterectomy prior to the onset of labor [conducted for reasons of placenta previa (n = 3) and placenta accreta (n = 1)]. Informed consent in writing for the use of tissue was obtained from the women undergoing surgery according to a protocol approved by the Institutional Review Board for Human Experimentation (University of Texas Southwestern).
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RESULTS |
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The minimum telokin promoter directs smooth muscle-specific
expression in transgenic mice.
Previous studies of telokin promoter-luciferase reporter genes in A10
smooth muscle cells demonstrated that a 310-bp fragment of the telokin
promoter extending from 163 to +147 is sufficient for maximal
cell-specific promoter activity (8). In this investigation, transgenic
mice were generated in which the 310-bp promoter fragment was fused to
SV40 large T antigen to determine whether this minimal promoter is
sufficient to mediate smooth muscle-specific expression in vivo.
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Telokin expression correlates with estrogen levels in human uterine smooth muscle. The finding of differential expression of the 310-bp transgene in uterine and intestinal smooth muscle, together with a previous report suggesting that telokin expression is stimulated by estrogen in chicken oviduct (26), prompted us to determine whether telokin expression in mammalian uterus is hormonally regulated. We analyzed telokin protein levels in myometrial tissues obtained from nonpregnant women during various phases of the menstrual cycle and from pregnant women at term (Fig. 4A). Telokin expression levels are significantly increased in myometrium obtained during pregnancy compared with tissues from nonpregnant women (2- to 3-fold; P < 0.02). Whereas telokin is expressed at uniformly high levels in the myometrium of pregnant women (Fig. 4A, lanes 1-4), the expression of telokin is variable in myometrium from nonpregnant women (Fig. 4A, lanes 5-8). In contrast, calponin is expressed at similar levels in the myometrium of both pregnant and nonpregnant individuals (Fig. 4A, bottom). We considered the possibility that the variable levels of telokin expression in the myometrium of nonpregnant women may reflect the hormonal milieu from which each sample was obtained. Tissues analyzed in Fig. 4, lanes 5 and 8, were obtained from women in the luteal phase of the menstrual cycle, the tissue in lane 6 was obtained during the follicular phase (day 12), and the sample analyzed in lane 7 was obtained from a woman treated with oral contraceptives (35 µg ethinyl estradiol and 1 mg norethindrone acetate). Endometrial histology confirmed the menstrual dating. Thus telokin expression is low during the progesterone-dominant natural luteal phase (lanes 5 and 8), and its expression is increased in the estrogen-dominant follicular phase (lane 6), during treatment with synthetic potent estrogens (lane 7), or during pregnancy (lanes 1-4). Conversely, the expression of telokin was significantly decreased (P < 0.02) in uterine tissues obtained from postmenopausal women (Fig. 4B, lanes 1-3) and from premenopausal women rendered hypoestrogenic with gonadotropin-releasing hormone analogs (Fig. 4B, lanes 6 and 7) compared with uterine tissues obtained from premenopausal women during the follicular phase of the menstrual cycle (Fig. 4B, lanes 3 and 4). These data suggest that estrogen may stimulate telokin expression in human uterine smooth muscle.
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The telokin promoter is estrogen responsive.
To determine whether deletion of an ERE could account for the
differences in the expression of 2.4-kb and 310-bp transgenes, experiments were performed to directly determine the responsiveness of
2.4-kb and 310-bp reporter genes to estradiol. T2.4 and T310 telokin-luciferase reporter genes (8) were transfected into A10 cells
either together with an estrogen receptor cDNA expression plasmid
[CMV-ER (14)] or with vector alone (CMV). After
transfection, cells were treated with
108 M 17
-estradiol or
vehicle, as indicated, for 24 h prior to analysis of reporter gene
activity. Estradiol resulted in a threefold stimulation of luciferase
activity driven by the 2.4-kb promoter (
2250 to +147; Fig.
5). No stimulation was observed in the
absence of transfected receptor (Fig. 5), suggesting that A10 cells
lack any endogenous receptor and that the stimulation of reporter gene
activity is estrogen receptor dependent. The estrogen stimulation was
saturable, with a maximal effect at
10
8 M estradiol (data not
shown). In contrast, the luciferase reporter gene driven by a 310-bp
rabbit telokin promoter fragment (
163 to +147) was not
stimulated by estradiol (Fig. 5). These results suggest that an ERE is
located between
163 and
2250 bp in the rabbit telokin
promoter.
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Identification of an ERE in the rabbit telokin promoter.
Sequence analysis of the rabbit telokin promoter between 2250
and
163 for steroid response elements revealed the presence of
three potential ERE half sites between
1474 and
1447
(Fig. 6A); no
glucocorticoid or progesterone response elements were found. Two of the
ERE half sites comprise an inverted palindrome with seven bp between
them. The third, most proximal, half site is a direct repeat of
the second separated by six nucleotides. A comparison of the telokin
ERE sequence and a consensus ERE is shown in Fig.
6A.
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DISCUSSION |
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Results indicate that a 310-bp minimal rabbit telokin promoter fragment is sufficient to mediate smooth muscle cell-specific expression of an SV40 large T-antigen transgene in vivo in transgenic mice. High levels of transgene expression were observed in smooth muscles of the digestive tract, urinary tract, and male reproductive tracts, and lower levels of expression were observed in vascular, airway, and uterine smooth muscle.
The expression of telokin closely resembles that of smooth muscle
-actin, with high levels of expression in visceral smooth muscle and
low levels of expression in vascular smooth muscle. It would thus seem
reasonable to propose that expression of these two genes is similarly
regulated. However, analyses of the
-actin and telokin promoters in
transgenic mice have revealed some fundamental differences in the
organization of important regulatory elements (8, 24). The regulatory
elements required to direct the appropriate smooth muscle-specific
expression of the smooth muscle
-actin gene are contained within an
11.6-kb fragment of the gene, including 2.8 kb of upstream sequence,
exon 1, intron 1, and a portion of exon 2. In contrast, the regulatory
elements required for smooth muscle-specific expression of telokin are
contained within a 310-bp proximal promoter fragment extending from
163 to +147. These data suggest that, although the pattern of
expression of telokin and smooth muscle
-actin is very similar, the
cis-acting regulatory elements
controlling the expression of these genes are organized in a very
different spatial manner. Identification of the regulatory elements
important for the expression of the
-actin gene will be required to
determine whether the two genes utilize similar elements that are
arranged differently or whether the two genes utilize distinct
cis-acting regulatory elements.
Comparison of the promoter and regulatory regions of the telokin gene
with those found in other smooth muscle-specific genes has thus far not
revealed any DNA binding activities that act as master regulators of
all smooth muscle-specific genes. In the telokin promoter (9), smooth
muscle -actin promoter (2, 21, 22), smooth muscle myosin heavy chain
promoter (17), and SM22
promoter (13), SRF binding to CArG elements
has been shown to be important for transcriptional activity. CArG
elements are also present in the smooth muscle
-actin (24) promoter. As SRF is ubiquitously expressed, it alone cannot account for the cell
specificity of these promoters. However, it remains possible that SRF
interacts with other cell-restricted factors to mediate smooth
muscle-specific gene expression.
SV40 large T-antigen expression driven by either 2.4-kb (8) or 310-bp
telokin promoter fragments in transgenic mice exhibited essentially
identical patterns of expression in all tissues except for uterus.
Although the 2.4-kb and 310-bp promoters were able to direct comparable
levels of transgene expression in intestinal smooth muscle, only the
2.4-kb fragment was able to direct high levels of transgene expression
in uterine smooth muscle (Fig. 3). These data suggest that a positively
acting element located between 2250 and
163 is required
for high levels of telokin expression in uterine but not intestinal
smooth muscle. These results also imply that in intestinal smooth
muscle there are transcription factors that bind to the 310-bp proximal
telokin promoter that are either absent or expressed at lower levels in uterine smooth muscle. Conversely, the expression pattern of SM22
promoter transgenes [i.e., high levels in vascular smooth muscle but not in visceral smooth muscle (16)] suggests that factors that bind the proximal SM22
promoter are present in vascular smooth
muscle cells but not visceral smooth muscle cells. Together these data
suggest that the regulation of gene expression in smooth muscle is
likely to be complex, with different smooth muscle tissues containing
distinct transcription factors or perhaps different amounts of common
factors. As a result, distinct
cis-acting regulatory elements within
a single gene are required for expression in different smooth muscle
types.
Analysis of telokin expression in human myometrium suggests that telokin expression may be regulated by estrogen in human uterine smooth muscle. The changes in levels of expression are specific to telokin and do not reflect general changes in smooth muscle contractile protein expression. For example, caponin (another smooth muscle-specific protein) is expressed at similar levels in all the human uterine samples examined (Fig. 4, A and B, bottom), and myosin light chain kinase and phosphatase activities are similar in myometrium from pregnant and nonpregnant women (28). It has recently been shown that telokin inhibits the phosphorylation of myosin by myosin light chain kinase by increasing the Michaelis-Menten constant for the myosin regulatory light chain (26). As a result, an increase in the amount of telokin relative to the kinase would be anticipated to decrease myosin light chain phosphorylation levels in response to a given stimulus. The hormonal regulation of telokin expression in human myometrial smooth muscle may thus play an important physiological role in regulating myometrial contractility. However, two observations suggest that the regulation of telokin expression by estrogen may be species dependent. For example, we observed no significant effect of estradiol on telokin expression levels in the uteri of ovariectomized rats (Herring and Bigsby, unpublished observation). In addition, although we were able to show that the rabbit telokin promoter was stimulated two- to threefold by estradiol in the presence of transfected estrogen receptor (Figs. 5 and 6), in parallel experiments the mouse telokin promoter (3.6-kb fragment) was not stimulated by estradiol (data not shown). Together, these data suggest that the estrogen regulation of telokin expression exhibited in rabbits and humans may not occur in rodents.
Results derived from reporter gene assays in A10 vascular smooth muscle
cells suggest that an unusual ERE between 1474 and
1447
mediates estrogen regulation of the rabbit telokin gene. The estrogen
receptor has been shown to bind to palindromic response elements with
the consensus aGGTCAnnnTGACCTt (where lowercase letters are not part of
the consensus) (29). In addition, a number of estrogen-responsive genes
contain imperfect palindromic EREs either in isolation or in multiple
arrays (4, 7). Imperfect repeats such as those found in the mouse
mammary tumor virus gene can synergize with other
cis-acting regulatory elements (19). Recently it has been shown that direct repeats of RGGTCA half sites can
also function as EREs. Interestingly, the space between the directly
repeated half sites is more flexible than that between palindromic
repeats. For example, direct repeats separated by four, five, or six
nucleotides have all been shown to bind to estrogen receptor (1, 11,
12, 19, 23). Direct repeats of ERE half elements bind estrogen receptor
approximately eightfold less efficiently than perfect palindromic
repeats and are thus functionally similar to imperfect palindromic
response elements. The ERE in the rabbit telokin gene is composed of
three half-sites, with the first two representing an inverted
palindromic consensus ERE separated by seven nucleotides; the third
half site is a direct repeat of the second half site separated by six
residues. Only the second half site has a purine residue at the
1 position that was shown to be important for estrogen receptor
binding to direct repeat elements (1). Despite these constraints, the
ERE in the rabbit telokin gene responds to estradiol with a level of stimulation in activity similar to that observed with a perfect palindromic ERE (Fig. 6). Synergy between the ERE and the proximal promoter may account for this stimulation and may mediate high levels
of telokin expression in uterine smooth muscle. In contrast, the
proximal promoter alone is sufficient to direct high levels of telokin
expression in intestinal smooth muscle.
In summary, the data presented demonstrate that a 310-bp fragment of
the telokin promoter extending from 163 to +147 is sufficient to
mediate smooth muscle-specific expression in transgenic mice. However,
high levels of transgene expression in uterine smooth muscle require
additional regulatory elements. One such element may include an ERE
located between
1474 and
1447. This ERE is required to
mediate estrogen stimulation of reporter gene activity in
cotransfection experiments. These data, together with data obtained
from analysis of SM22
and smooth muscle
-actin promoters in
transgenic mice, suggest that distinct regulatory elements within a
single gene are required for expression in different smooth muscle
tissues and that different genes may use distinct regulatory elements
for expression in a single smooth muscle tissue.
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ACKNOWLEDGEMENTS |
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This work was supported in part by Grant-In-Aid 95010980 from the American Heart Association (to B. P. Herring), National Institute of Child Health and Human Development Grant HD-11149 (to A. R. Word), and a postdoctoral fellowship from the American Heart Association Indiana Affiliate (to A. F. Smith).
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FOOTNOTES |
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Address for reprint requests: B. P. Herring, Dept. of Physiology and Biophysics, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5120.
Received 16 July 1997; accepted in final form 31 December 1997.
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