EDITORIAL FOCUS
A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin

Aiping F. Smith1, Robert M. Bigsby2, R. Ann Word3, and B. Paul Herring1

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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 SM22alpha (16) and smooth muscle gamma -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 gamma -actin transgenes are expressed at high levels in visceral smooth muscle and at lower levels in vascular smooth muscle. In contrast, the mouse SM22alpha 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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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)-beta 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 17beta -estradiol (10-8 M) or vehicle. Cell extracts (200 µl/well) were prepared for luciferase and beta -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).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

Western blot analysis was used to compare the tissue distribution of T antigen to the distribution of the endogenous telokin protein in tissues obtained from several transgenic founder animals. Results from this analysis demonstrate that transgene expression directed by the 310-bp promoter fragment is almost identical to that directed by the 2.4-kb fragment. Both promoter fragments direct smooth muscle-specific expression of T antigen that mirrors endogenous telokin expression (Fig. 1). High levels of expression were observed in bladder and throughout the digestive tract, with lower levels of expression in lung and vascular smooth muscle. One notable difference between transgenes is that in all founder animals the 310-bp transgene was expressed at low to undetectable levels in uterus (Figs. 1 and 2). Comparison of T-antigen expression driven by either the 2.4-kb or 310-bp promoter fragments in uterus and colon of several founder animals demonstrates that, although both transgenes were expressed at similar levels in smooth muscle of the colon, only the transgene driven by the 2.4-kb promoter was expressed at high levels in uterine smooth muscle (Fig. 2). These data suggest that an additional regulatory element located between -163 and -2250 is required for high levels of transgene expression in uterine smooth muscle.


View larger version (68K):
[in this window]
[in a new window]
 
Fig. 1.   Telokin-T-antigen expression in transgenic mice is restricted to smooth muscle tissues. Western blot analysis of various tissue extracts (25 µg) from telokin 2.4-kb promoter fragment (T2.4)-T-antigen (A) and telokin 310-bp promoter fragment (T310)-T- antigen (B) transgenic mice. AT2, extracts from a simian virus 40 (SV40) large T-antigen-transformed cardiac cell line used as a positive control for T-antigen expression. Blots were reacted with a polyclonal antibody to SV40 large T antigen (top blot of each pair) or with a polyclonal antibody to telokin (bottom blot of each pair). TAG and TEL, positions of bands corresponding to T antigen and telokin, respectively; SKEL, gastrocnemius muscle. Positions of molecular mass markers are indicated in kDa at left.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 2.   T310-T-antigen transgene is expressed at different levels in uterus and colon. Western blot analysis of T-antigen transgene expression and endogenous telokin expression in tissue extracts (25 µg) obtained from uterus and colon of two T2.4-T-antigen (T2.4, A and B) and three T310-T-antigen (T310, A-C) founder animals. Blots were reacted with a polyclonal antibody to SV40 large T antigen (top) or with a polyclonal antibody to telokin (bottom). Positions of molecular mass markers are indicated in kDa at left.

To confirm that T antigen detected in smooth muscle tissues by Western blotting was restricted to smooth muscle cells, T-antigen localization was examined by immunofluorescence analysis of tissue sections. In all tissues examined, T-antigen expression is restricted to the nuclei of smooth muscle cells (Fig. 3).


View larger version (90K):
[in this window]
[in a new window]
 
Fig. 3.   T310-T-antigen transgene expression is restricted to smooth muscle cells. Cryosections 8 µm thick were cut from various frozen tissues samples obtained from adult transgenic mice. T-antigen expression was detected using a polyclonal antibody to T antigen and visualized using fluorescein conjugated to donkey anti-rabbit IgG (green). Sections were counterstained with nuclear stain Hoechst and visualized by epifluorescence (blue). Representative sections from kidney (A and B), skeletal muscle (C), cardiac muscle (D), colon (E and F), and ileum (G and H) are shown. Scale bars for each pair of panels, 50 µm.

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.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 4.   Telokin expression is increased in human myometrium during pregnancy. Western blot analysis of telokin (top) and calponin (bottom) expression in human myometrial tissues (15 µg extract/lane). Blots were reacted with a polyclonal antibody to telokin (top) (6) or with a monoclonal antibody specific for smooth muscle calponin (bottom; Sigma, clone hcp). Positions of molecular mass standards (kDa) are indicated at left. A: samples obtained from pregnant (lanes 1-4) and nonpregnant (lanes 5-8) women were analyzed. Samples analyzed in lanes 5 and 8 were obtained from women in luteal phase of menstrual cycle (days 21 and 16, respectively). Sample in lane 6 was obtained during follicular phase (day 12). Sample analyzed in lane 7 was obtained from a woman treated with oral contraceptives. B: samples obtained from postmenopausal women (POST-M; lanes 1-3), premenopausal women in follicular phase of menstrual cycle (FOL; lanes 4 and 5), and premenopausal women rendered hypoestrogenic with a gonadotropin-releasing hormone (GnRH) analog (Lupron, 37.5 mg/mo) for 2 (lane 6) and 4 (lane 7) mo.

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 10-8 M 17beta -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.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5.   Telokin promoter reporter genes are stimulated by estradiol. A10 vascular smooth muscle cells were transfected with T2.4-pGL2-Basic (solid bars) and T310-pGL2-Basic (hatched bars) telokin promoter-luciferase reporter genes together with an estrogen receptor (ER) expression vector (+) or with vector alone (-) and a beta -galactosidase internal control plasmid, as described in MATERIALS AND METHODS. Transfected cells were treated with 10-8 M estradiol (ES; +) or vehicle (-) for 24 h; extracts were then prepared for luciferase and beta -galactosidase assays. Luciferase activities (means ± SE, n = 6), normalized to beta -galactosidase, are expressed as a percentage of activity obtained from T2.4-pGL2-Basic luciferase plasmid in absence of both transfected receptor and estradiol.

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.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 6.   Identification of an estrogen response element (ERE) in telokin promoter. A: nucleotide sequence of telokin ERE compared with a consensus (Cons) ERE; 3 consensus ERE half sites are shown in uppercase. B: luciferase activity, normalized to a beta -galactosidase internal control, obtained from A10 cells transfected with luciferase plasmids indicated at bottom. Data are means ± SE of 6-11 independent transfections. Cells were treated with 10-8 M estradiol (solid bars) or vehicle (hatched bars) for 24 h. Activities are expressed as a percentage of vehicle-treated level. T2.4, wild-type 2.4-kb fragment (-2250 to +147) of telokin promoter; TEREDelta , 2.4-kb fragment of promoter in which ERE (-1492 to -1421) has been deleted; T310, 310-bp fragment of promoter; TERE T310, 310-bp fragment of promoter to which telokin ERE (shown in A) has been added 5' to promoter; ERE-TK, minimal thymidine kinase promoter (-109 to +52) containing a consensus palindromic ERE (shown in A) at -109.

Two strategies were used to determine whether the ERE identified by sequence analysis is responsible for mediating the estrogen stimulation of the 2.4-kb telokin promoter. First, a luciferase fusion gene was generated in which the ERE was deleted (TEREDelta ), and, second, a DNA fragment containing the putative ERE (Fig. 6A) was ligated 5' to the minimal 310-bp promoter (TERE-T310). These constructs, together with CMV-ER, were transfected into A10 smooth muscle cells, and luciferase activity was determined in the presence or absence of 17beta -estradiol. Deletion of the ERE-like sequence in the 2.4-kb promoter (-1474 to -1447) abolished estrogen stimulation of 2.4-kb reporter gene activity (Fig. 6B). Conversely, addition of this ERE sequence to the 310-bp reporter gene conferred estrogen responsiveness, with estradiol stimulating promoter activity threefold (TERE-T310; Fig. 6B). A reporter gene containing the minimal thymidine kinase promoter and a consensus ERE (cERE-TK) was stimulated twofold by estradiol under these experimental conditions. These results show that the telokin gene is regulated by an ERE between -1474 and -1447.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 gamma -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 gamma -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 gamma -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 gamma -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 gamma -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 alpha -actin promoter (2, 21, 22), smooth muscle myosin heavy chain promoter (17), and SM22alpha 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 gamma -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 SM22alpha promoter transgenes [i.e., high levels in vascular smooth muscle but not in visceral smooth muscle (16)] suggests that factors that bind the proximal SM22alpha 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 SM22alpha and smooth muscle gamma -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.

    ACKNOWLEDGEMENTS

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).

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Aumais, J. P., H. S. Lee, C. DeGannes, J. Horsford, and J. H. White. Function of directly repeated half-sites as response elements for steroid hormone receptors. J. Biol. Chem. 271: 12568-12577, 1996[Abstract/Free Full Text].

2.   Blank, R. S., T. C. McQuinn, K. C. Yin, M. M. Thompson, K. Takeyasu, R. J. Schwartz, and G. K. Owens. Elements of the smooth muscle alpha-actin promoter required in cis for transcriptional activation in smooth muscle. Evidence for cell type-specific regulation. J. Biol. Chem. 267: 984-989, 1992[Abstract/Free Full Text].

3.   Chamley-Campbell, J. H., and G. R. Campbell. What controls smooth muscle phenotype. Atherosclerosis 40: 347-357, 1981[Medline].

4.   Chang, T.-C., A. M. Nardulli, D. Lew, and D. J. Shapiro. The role of estrogen response elements in expression of the Xenopus laevis vitellogenin B1 gene. Mol. Endocrinol. 6: 346-354, 1992[Abstract].

5.   Field, L. J. Atrial natriuretic factor-SV40 T antigen transgenes produce cardiac tumors and cardiac arrhythmias in mice. Science 239: 1029-1033, 1988[Medline].

6.   Gallagher, P. J., and B. P. Herring. The carboxyl terminus of the smooth muscle myosin light chain kinase is expressed as an independent protein, telokin. J. Biol. Chem. 266: 23945-23952, 1991[Abstract/Free Full Text].

7.   Green, S., and P. Chambon. The oestrogen receptor: from perception to mechanism. In: Nuclear Hormone Receptors: Molecular Mechanisms, Cellular Functions, Clinical Abnormalities, edited by M. G. Parker. San Diego, CA: Academic, 1991, p. 15-38.

8.   Herring, B. P., and A. F. Smith. Telokin expression is mediated by a smooth muscle cell-specific promoter. Am. J. Physiol. 270 (Cell Physiol. 39): C1656-C1665, 1996[Abstract/Free Full Text].

9.   Herring, B. P., and A. F. Smith. Telokin expression in A10 smooth muscle cells requires serum response factor. Am. J. Physiol. 272 (Cell Physiol. 41): C1394-C1404, 1997[Abstract/Free Full Text].

10.   Katayama, E., G. Scott-Woo, and M. Ikebe. Effect of caldesmon on the assembly of smooth muscle myosin. J. Biol. Chem. 270: 3919-3925, 1995[Abstract/Free Full Text].

11.   Kato, S., H. Sasaki, M. Suzawa, S. Masushige, L. Tora, P. Chambon, and H. Gronemeyer. Widely spaced, directly repeated PuGGTCA elements act as promiscuous enhancers for different classes of nuclear receptors. Mol. Cell. Biol. 15: 5858-5867, 1995[Abstract].

12.   Kato, S., L. Tora, J. Yamauchi, S. Masushige, M. Bellard, and P. Chambon. A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5'-TGACC-3' motifs acting synergistically. Cell 68: 731-742, 1992[Medline].

13.   Kim, S., H. S. Ip, M. M. Lu, C. Clendenin, and M. S. Parmacek. A serum response factor-dependent transcriptional regulatory program identifies distinct smooth muscle cell sublineages. Mol. Cell Biol. 17: 2266-2278, 1997[Abstract].

14.   Kumar, V., S. Green, G. Stack, M. Berry, J.-R. Jin, and P. Chambon. Functional domains of the human estrogen receptor. Cell 51: 941-951, 1987[Medline].

15.   Kunkel, T. A., J. D. Roberts, and R. A. Zakour. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154: 367-382, 1987[Medline].

16.   Li, L., J. M. Miano, B. Mercer, and E. N. Olson. Expression of the SM22alpha promoter in transgenic mice provides evidence for distinct transcriptional regulatory programs in vascular and visceral smooth muscle cells. J. Cell Biol. 132: 849-859, 1996[Abstract].

17.   Madsen, C. S., J. C. Hershey, M. B. Hautmann, S. L. White, and G. K. Owens. Expression of the smooth muscle myosin heavy chain gene is regulated by a negative-acting GC-rich element located between two positive-acting serum response factor-binding elements. J. Biol. Chem. 272: 6332-6340, 1997[Abstract/Free Full Text].

18.   Majesky, M. W., C. M. Giachelli, M. A. Reidy, and S. M. Schwartz. Rat carotid neointimal smooth muscle cells reexpress a developmentally regulated mRNA phenotype during repair of arterial injury. Circ. Res. 71: 759-768, 1992[Abstract].

19.   Martinez, E., and W. Wahli. Characterization of hormone response elements. In: Nuclear Hormone Receptors: Molecular Mechanisms, Cellular Functions, Clinical Abnormalities, edited by M. G. Parker. San Diego, CA: Academic, 1991, p. 125-153.

20.   Meyer, T., R. Koop, E. von Angerer, H. Schonenberger, and E. Holler. A rapid luciferase transfection assay for transcription activation effects and stability control of estrogenic drugs in cell cultures. J. Cancer Res. Clin. Oncol. 120: 359-364, 1994[Medline].

21.   Min, B., D. N. Foster, and A. R. Strauch. The 5'-flanking region of the mouse vascular smooth muscle alpha -actin gene contains evolutionarily conserved sequence motifs within a functional promoter. J. Biol. Chem. 265: 16667-16675, 1990[Abstract/Free Full Text].

22.   Nakano, Y., T. Nishihara, S. Sasayama, T. Miwa, S. Kamada, and T. Kakunaga. Transcriptional regulatory elements in the 5' upstream and first intron regions of the human smooth muscle (aortic type) alpha -actin gene. Gene 99: 285-289, 1991[Medline].

23.   Nawaz, Z., M. J. Tsai, D. P. McDonnell, and B. W. O'Malley. Identification of novel steroid-response elements. Gene Expr. 2: 39-47, 1992[Medline].

24.   Qian, J., A. Kumar, J. C. Szucsik, and J. L. Lessard. Tissue and developmental specific expression of murine smooth muscle. Dev. Dyn. 207: 135-144, 1996[Medline].

25.   Shirinsky, V. P., A. V. Vorotnikov, K. G. Birukov, A. K. Nanaev, M. Collinge, T. J. Lukas, J. R. Sellers, and D. M. Watterson. A kinase-related protein stabilizes unphosphorylated smooth muscle myosin minifilaments in the presence of ATP. J. Biol. Chem. 268: 16578-16583, 1993[Abstract/Free Full Text].

26.   Silver, D. L., A. V. Vorotnikov, D. M. Watterson, V. P. Shirinsky, and J. R. Sellers. Sites of interaction between kinase-related protein and smooth muscle myosin. J. Biol. Chem. 272: 25353-25359, 1997[Abstract/Free Full Text].

27.   Touyz, R., and E. L. Schiffrin. Biology of blood vessels in hypertension. Cardiol. Rev. 1: 87-96, 1993.

28.   Word, R. A., J. T. Stull, M. L. Casey, and K. E. Kamm. Contractile elements and myosin light chain phosphorylation in myometrial tissue from nonpregnant and pregnant women. J. Clin. Invest. 92: 29-37, 1993[Medline].

29.   Zilliacus, J., A. P. Wright, J. Carlstedt-Duke, and J. Gustafsson. Structural determinants of DNA-binding specificity by steroid receptors. Mol. Endocrinol. 9: 389-400, 1995[Medline].


AJP Cell Physiol 274(5):C1188-C1195
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society