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Originally published In Press as doi:10.1074/jbc.M313822200 on January 26, 2004

J. Biol. Chem., Vol. 279, Issue 16, 15929-15937, April 16, 2004
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Activation of the Smooth Muscle-specific Telokin Gene by Thyrotroph Embryonic Factor (TEF)*

Jiliang Zhou, April M. Hoggatt, and B. Paul Herring{ddagger}

From the Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120

Received for publication, December 17, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transcription of the telokin gene is restricted to smooth muscle cells throughout development, making this gene an excellent model for unraveling the mechanisms that regulate gene expression in smooth muscle tissues. To identify proteins that bind to the telokin promoter, the AT-rich/CArG core of the promoter was used as a probe to perform a Southwestern screen of a mouse bladder cDNA library. Four clones corresponding to two distinct isoforms of mouse thyrotroph embryonic factor (TEF{alpha} and TEF{beta}) were identified from this screen. The two TEF isoforms differ from each other at their amino termini and result from alternative promoter usage. An RNase protection assay showed that both TEF isoforms are expressed at high levels in mouse lung, bladder, kidney, gut, and brain. Gel mobility shift assays demonstrated that purified TEF protein can specifically bind to an AT-rich region within the core of the telokin promoter. Furthermore, when overexpressed in 10T1/2 cells, TEF significantly increased the activity of a telokin promoter-reporter gene; this activation was further augmented by elevated intracellular calcium levels. In contrast, overexpression of TEF had no effect on reporter genes driven by SM22{alpha}, smooth muscle {alpha}-actin, or smooth muscle myosin heavy chain promoters. Consistent with these results, overexpression of TEF{alpha} and TEF{beta} in A10 cells, using adenoviral vectors, increased expression of endogenous telokin without altering expression of myosin light chain 20, SM22{alpha}, smooth muscle {alpha}-actin, or calponin. These findings suggest that TEF factors contribute to the activation of the telokin promoter in smooth muscle cells in a calcium-dependent manner. These data also suggest that distinct transcription factors are required to control the expression of different smooth muscle genes in a single tissue.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is extensive evidence showing that altered control of the differentiated state of smooth muscle cells contributes to the development and/or progression of a variety of diseases, including atherosclerosis, hypertension, and asthma. These diseases are all associated with decreased expression of proteins required for the differentiated function of the smooth muscle cells. An understanding of the mechanisms that control smooth muscle cell differentiation is required before it will be possible to determine how these control processes are altered in pathological conditions.

To begin to elucidate the molecular mechanisms that control smooth muscle cell differentiation, we have sought to determine which transcription factors are important for regulating expression of the telokin gene in smooth muscle tissues. Telokin is a smooth muscle-restricted protein with an amino acid sequence that is identical to the carboxyl-terminal domain of myosin light chain kinase (MLCK)1 (1). Although the physiological function of telokin has not been fully elucidated, previous studies have suggested that telokin may play a role in regulating smooth muscle contractility. Telokin has been shown to stabilize unphosphorylated myosin filaments in vitro (2, 3). Telokin has also been shown to accelerate the dephosphorylation of myosin light chain and induce relaxation of permeabilized smooth muscle strips through activation of myosin light chain phosphatase (4).

Transcription of telokin is strictly restricted to smooth muscle cells throughout mouse development, which makes this gene a good marker for studying the control of smooth muscle cell lineage (5). Telokin mRNA is transcribed from an internal promoter, located within an intron, in the 3' region of the MLCK gene (6). In vitro reporter gene assays have shown that 310 bp (–163 to +147) and 370 bp (–190 to +180) fragments of the rabbit and mouse telokin promoters, respectively, are sufficient to mediate cell-specific expression (6, 7). Both of these telokin promoters are also sufficient to direct transgene expression specifically to smooth muscle tissues in adult mice (7, 8). Transgenes driven by these promoters are expressed at much higher levels in visceral as opposed to vascular smooth muscle tissues. Several positive-acting elements within the minimal telokin promoter, including an E box, AT-rich region, and CArG box, were shown to be important for reporter gene activity in A10 smooth muscle cells (9). A core fragment of the minimal telokin promoter, including the E box, AT-rich region, and CArG box, has been shown to specifically increase expression of the normally vascular smooth muscle-specific SM22{alpha} promoter in smooth muscle cells of the bladder (7). The AT-rich region acting together with the CArG box appears to be important for determining cell specificity because these combined elements increase the activity of a minimal thymidine kinase promoter in A10 smooth muscle cells but not in fibroblasts. In contrast, the AT-rich element alone had no effect on thymidine kinase promoter activity and the CArG element increased activity of the promoter in both fibroblasts and smooth muscle cells (7).

The CArG element in the telokin promoter has been shown to bind to serum response factor (SRF) (9). Many smooth muscle-specific genes require critical evolutionarily conserved CArG boxes that bind SRF for their expression in smooth muscle cells in vitro and in transgenic mice (10). Recent studies have demonstrated that the interaction of SRF with the co-activator myocardin is a critical determinant of smooth muscle development (11). Although interaction of SRF with myocardin is clearly critical for activating most smooth muscle-restricted genes, neither myocardin nor SRF is expressed only in smooth muscle cells; hence, additional regulatory proteins must be required to restrict gene expression to smooth muscle cells.

A number of studies have suggested that AT-rich regions are involved in the regulation of expression of smooth muscle-restricted genes. Modulator recognition factor 2, a member of the AT-rich interaction domain family of transcription factors, has been shown to induce the pluripotent neural crest cell line (MONC-1) to differentiate into smooth muscle cells (12). We previously identified a transcription factor of the forkhead family, Foxq1 (hepatocyte nuclear factor-3 homologue 1) that binds to the AT-rich region of the telokin promoter and strongly represses telokin promoter activity when overexpressed in A10 vascular smooth muscle cells (13). In the current study we employed a lambda Southwestern screen of a mouse bladder cDNA library, using the AT-rich/CArG core of the telokin promoter as a probe to identify additional proteins that can bind to this region of the promoter. Four clones corresponding to two distinct isoforms of mouse thyrotroph enhancer factor (TEF) were identified (14). The two TEF isoforms differ from each other at their amino termini and arise from alternative promoter usage. Gel mobility shift assays confirmed that purified TEF protein specifically bound to the AT-rich region (TTATATAA) within the core of the telokin promoter. Furthermore, TEF can significantly increase telokin promoter-reporter gene activity in 10T1/2 cells in a calcium-dependent manner. No activation of other smooth muscle promoters such as SM22{alpha}, smooth muscle {alpha}-actin (SM {alpha}-actin), and smooth muscle myosin heavy chain (SM-MHC) was observed. Overexpression of TEF{alpha} and TEF{beta} in A10 cells up-regulated expression of endogenous telokin but not SM22{alpha}, SM {alpha}-actin, and SM-MHC expression. These data suggest that TEF may play an important role in the activation of telokin expression in smooth muscle tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Lambda Southwestern Screen—A mouse bladder cDNA library in lambda gt11, described previously (13), was screened to identify LAC·cDNA-encoded fusion proteins that were capable of binding to the AT-rich/CArG core of the telokin promoter. Escherichia coli Y1090r-were infected with the lambda gt11 cDNA library phage and plated on 150-mm dishes using standard procedures (15). Plates were overlaid with nitrocellulose filters soaked in 10 mM isopropyl-1-thio-{beta}-D-galactopyranoside to induce expression of LAC fusion proteins. After 6 h the filters were removed, blocked by incubation in BLOTTO (5% nonfat milk powder, 50 mM Tris·Cl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol) for 1 h at room temperature. Filters were then washed three times in binding buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol) and stored overnight at 4 °C. Filters were incubated with probes at a concentration of 106 cpm/ml in binding buffer for 1 h at room temperature. Unbound probe was then removed by 4 x 7.5-min washes with binding buffer. Positive clones were then identified by autoradiography. The probe used for this screen comprised three tandem copies of the core of the mouse telokin promoter (–90 to –53) as described previously (13). This fragment was generated by ligating double-stranded oligonucleotides into EcoR1-linearized pGEM7Z (Promega). The sequences of these oligonucleotides were sense 5'-AATTCTGCAGTTGCTTTATATAAACTATCCCTTTTATGGGAGC-3' and antisense 5'-AATTGCTCCCATAAAAGGGATAGTTTATATAAAGCAACTGCAG-3'. The sequence and orientation of the fragments was confirmed by direct DNA sequencing. The trimerized fragment was isolated by digestion with XhoI and BamHI, separated by agarose gel electrophoresis, and purified using Qiaex II beads (Qiagen, Valencia, CA). This fragment was then end-labeled using the Klenow fragment of DNA polymerase and 32P(dCTP), and unincorporated nucleotide was removed by spin column chromatography (Bio-Rad). Approximately 1 x 106 plaques were screened from the library; positive plaques were picked and rescreened until clonal. DNA was prepared from positive plaques using lamdabsorb according to the manufacturer's directions (Promega). cDNAs were then subcloned into pGEM5Z subjected to direct automated DNA sequencing (Seqwright, Houston, TX).

RNase Protection Assays—Total RNA was isolated from tissues and cells using guanidinium isothiocyanate (15). A 260-bp fragment of the TEF{beta} cDNA (corresponding to nucleotides 1–260) was subcloned into pGEM7Z (Promega). The plasmid was linearized with SalI and a 32P-labeled antisense riboprobe (300 bp) generated using SP6 polymerase and a Maxi Script in vitro transcription kit according to the manufacturer's directions (Ambion, Austin, TX). The TEF riboprobe was gelpurified on a 6% polyacrylamide/8 M urea gel and eluted overnight at 37 °C. Ribonuclease protection assays were then performed according to the manufacturer's directions (Standard RPA II kit; Ambion). Briefly, 1 x 105 cpm of gel-purified TEF riboprobe was co-precipitated with 20 µg of RNA and hybridized overnight at 42 °C. Samples were digested with RNase A/T1 at 1:100 dilution for 30 min at 37 °C and then inactivated and precipitated. Samples were solubilized in 8 µl of gel loading buffer. One-half the volume was loaded onto a 6% polyacrylamide/8 M urea gel run at 55 W for 2 h. Riboprobes of known sizes were run alongside RPA samples to verify the size of the probe and protected fragments. The riboprobe used will protect a 260-bp fragment of mouse TEF{beta} but only a 131-bp fragment of TEF{alpha}.

Gel Mobility Shift Analysis of DNA Binding—Mobility shift assays were performed in a final volume of 15 µl. Binding mixes contained 0.2 ng (1.5 x 104 cpm) of end-labeled double-stranded DNA probe, 200 ng of poly(dI-dC), 4.5 µg of bovine serum albumin, and various amounts of purified recombinant protein as indicated in a binding buffer containing 12 mM HEPES, pH 7.9, 60 mM KCl, 4 mM MgCl2, 10% glycerol, 1 mM dithiothreitol. All binding reactions were incubated for 15 min at room temperature followed by 1 h on ice except where indicated. For supershift assays, 1 µl of antibody was added to the binding assay mix after this initial incubation and then subsequently incubated on ice for a further 1 h. Polyclonal antibody to His6 was obtained from Clontech (Palo Alto, CA). A 50-fold excess of unlabeled double-stranded competitors was included in some reactions as indicated in the figure legends. The sequences of the sense strand of each probe were: AT/CArG, 5'-GCTTTATATAAACTATCCCTTTTATGGGAGCT-3'; AT, 5'-CAGCCTGCAGTTGTTTATATAAACTATCC-3'; CArG, 5'-CTATCCCTTTTATGGGAGCTGAAG-3'. Additional oligonucleotides used in competition assays were: E box, 5'-CGGAGCTGTCTCAGCCTGCAGTTGCTTTA-3', and AP2, 5'-CCGATCGAACTGACCGCCCGCGGCCCGT-3'. The cold AT MUT sequence was identical as AT probe except both underlined thymine residues were mutated to cytosines. Annealed oligonucleotides were labeled using [{alpha}-32P]dCTP and Klenow DNA polymerase (Promega). Unincorporated [{alpha}-32P]dCTP was removed by agarose gel electrophoresis. The DNA·protein complexes were resolved by electrophoresis through 4% polyacrylamide gels containing 6.75 mM Tris (pH 7.9), 3.3 mM sodium acetate (pH 7.9), 1 mM EDTA, and 2.5% glycerol. The gel was dried and autoradiographed with intensifying screens at –80 °C overnight.

Expression of Recombinant Proteins in Bacteria—Full-length human SRF, mouse TEF{alpha}, and TEF-DN (dominant negative) were expressed in bacteria using the pET expression system (Novagen, EMD Biosciences San Diego, CA). SRF and TEF{alpha} were purified as described previously (13). TEF-DN was used as an unpurified bacterial lysate. Mammalian Expression and Reporter Gene Assays—For expression in mammalian cells, TEF cDNAs were cloned into pcDNA 3.1 HisC (Invitrogen). This resulted in the expression of TEF as a fusion protein with amino-terminal His6 and Omni epitope tags. The resultant plasmids were sequenced to verify the integrity of the insert.

All promoter reporter genes were constructed by cloning fragments of promoters into the pGL2B luciferase vector except for smooth muscle {alpha}-actin and myosin heavy chain promoters, which are in the pGL3B vector (Promega). The rabbit telokin promoter-luciferase reporter gene used includes nucleotides –256 to +147 of the telokin gene as described previously (6). The SM22{alpha}-luciferase reporter gene includes nucleotides –475 to +61 of mouse SM22{alpha} (16, 17). The SM {alpha}-actin {alpha} promoter fragment extended from nucleotide –2,555 to +2,813 (18) and the smooth muscle myosin heavy chain promoter from –4,200 to +11,600 (19). The minimal TK promoter used comprised nucleotides –113 to +20 of the thymidine kinase gene. Point mutations in the rabbit telokin promoter were generated as described previously (9). Plasmids were transfected into rat A10 smooth muscle cells or mouse 10T1/2 fibroblasts cells using FuGENE6 (Roche Applied Science). A10 smooth muscle cells were grown in high-glucose Dulbecco's modified Eagle's medium containing 50 units/ml penicillin, 50 mg/ml streptomycin, and 20% fetal bovine serum. 10T1/2 fibroblast cells and COS cells were grown in medium supplemented with 10% fetal bovine serum. A10 and 10T1/2 cells to be transfected were seeded at 3 x 104 cells/well in 24-well plates. 16–18 h post-seeding, each dish was washed once with phosphate-buffered saline (pH 7.4), replaced with 0.5 ml of complete medium, incubated with a total of 1 µg of plasmid DNA (0.5 µg of promoter-luciferase plasmid, 0.25 µg of expression plasmid, and 0.25 µg of pRLminTK-luciferase plasmid as an internal control) and 2 µl of FuGENE in 50 µl of Dulbecco's modified Eagle's medium. 24 h later, extracts (100 µl/well) were prepared for measurement of luciferase activity. The level of promoter activity was evaluated by determining the level of firefly luciferase activity relative to the control Renilla luciferase, using the dual luciferase assay system essentially as described by the manufacturer (Promega). A minimum of six independent transfections was performed, and all assays were replicated at least twice. Results are reported as the mean ± S.E. All variables were analyzed by t test, and significance was set at p <=0.05. For verification of expression of wild type TEF and dominant negative mutant TEF protein, 24 h after transfection COS cell lysates were generated using RIPA buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 2 mM EDTA, 0.01 M sodium phosphate, pH 7.2) containing protease inhibitors and analyzed by Western blotting as described below.

Adenovirus Construction and Cell Infection—Adenovirus constructs were made using the adeno-X vectors obtained from Clontech, essentially following the manufacturer's instructions (BD Biosciences). In brief, the TEF full-length protein coding region or dominant negative mutant lacking the transcription activation domain was amplified by polymerase chain reaction (PCR) subcloned to the pShuttle vector and transferred into the adeno-X genome. The recombinant adenovirus was packaged in HEK293 cells and amplified to obtain high titer stocks. For adenoviral infection, A10 cells or 10T1/2 cells were seeded in 6-well plates at a density of 2 x 105 cells/well and grown overnight to near confluence. These cells were washed with phosphate-buffered saline to remove serum and infected with adenovirus encoding LacZ, TEF, or TEF-DN in phosphate-buffered saline at a multiplicity of infection of 100 for 4 h at 37 °C. These conditions resulted in close to 100% infection of cells. 72 h following infection, cell protein extracts were prepared using RIPA buffer and protein concentrations were determined using the BCA protein assay kit (Pierce).

Western Blotting—Western blotting analysis was carried out essentially as described previously (20). Fifteen micrograms of protein were fractionated on 7.5 or 15% SDS-polyacrylamide gels. The protein sample was electrophoretically transferred to a polyvinylidene difluoride membrane and verified by Ponceau S staining. The membrane was then probed with a series of antibodies. When required, before reacting with a subsequent antibody, the membrane was stripped and reprobed with second-step antibody to confirm that the previous antibody had been stripped from the blots. The secondary antibody, anti-mouse or anti-rabbit IgG (1:10,000 dilution), conjugated with horseradish peroxidase was visualized using Supersignal West Pico chemiluminescent substrate (Pierce) according to the manufacturer's instructions. Chemiluminescence was detected and quantitated using a CCD camera system (Fujifilm, Stamford, CT). Antibodies used in this study were: polyclonal antibody against telokin (1:6,000) (1), HA tag (1:1000; BabCO), LC20 (1:5,000; a gift from Dr. Patricia Gallagher), SM22{alpha} (1:6000; a gift from Dr. Len Adam), calponin (1:10,000; Sigma), SM {alpha}-actin (1:10,000; Sigma), SRF (1:10,000; Santa Cruz Biotechnology), and MLCK (1:10,000; Sigma).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Thyrotroph Embryonic Factor as a Telokin AT/CArG-binding Protein—Previously we have shown that the CArG box in the telokin promoter together with an adjacent AT-rich region are important for smooth muscle cell-selective expression of a reporter gene in vitro and in vivo (7). When this fragment was added to an arterial smooth muscle-selective SM22{alpha} transgene reporter, gene activity was selectively increased in bladder smooth muscle cells. This region is also very highly conserved across mammalian species, suggesting that important regulatory factors are likely to interact with this region. To identify transcription factors that can bind to the AT/CArG region of the telokin promoter, a Southwestern screen of a mouse bladder cDNA library was performed. From this screen four cDNAs were isolated that encoded fragments of TEF (14). The cDNAs isolated represent two distinct TEF isoforms homologous to isoforms TEF{alpha}/{alpha} and TEF{beta}/{alpha} (referred to as VBP in chicken) described previously in chicken (21) that differ from each other at their amino termini. Sequencing confirmed that the coding region of our TEF{alpha}/{alpha} clone was identical to a published mouse TEF cDNA sequence (GenBankTM accession number AF194420 [GenBank] ) (Fig. 1A). TEF is a member of the PAR (proline- and acidic-amino acid-rich) subfamily of bZIP transcription factors. Both isoforms have identical leucine zipper domains (bZip, amino acid 254–301), DNA-binding domains (DB, amino acids 216–254), PAR domains (PAR, amino acids 164–216), and transactivation domains (TA, amino acids 71–164). The only difference between the isoforms is that the first 49 amino acids of TEF{alpha} are distinct from the first 15 amino acids of TEF{beta} (Fig. 1B). These differences likely result from alternate promoter usage resulting in distinct first exons (Fig. 1C).



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  		FIG. 1.
Schematic summary of TEF{alpha} and -{beta} clones obtained from lambda Southwestern screen. A, schematic representation of the domain structures of TEF{alpha}, TEF{beta}, and TEF dominant negative proteins. The numbers above the schematic refer to the amino acids of TEF{alpha}. TA, transactivation domain; PAR, proline and acid-rich region; DB, DNA-binding domain; ZIP, leucine zipper domain. DN-TEF is generated by truncation of the TEF N-terminal transactivation domain. The unique amino-terminal regions of TEF{alpha} and -{beta} are checkered and striped, respectively. TEF clones identified from a Southwestern screen of a mouse cDNA expression library are aligned to the protein schematics. The clones are aligned to the published mouse TEF cDNA sequence (GenBankTM accession number AF194420 [GenBank] ). Three TEF{alpha} clones and a single TEF{beta} clone were identified. The overlapping sequences of the TEF{alpha} and -{beta} clones are identical except at the 5'-end, as indicated by the dashed line on the TEF{beta} sequence (GenBankTM accession number AY540632 [GenBank] ). A full-length TEF{alpha} cDNA sequence was generated by merging the sequences from the three overlapping clones (GenBankTM accession number AY540631 [GenBank] ). The position of the TEF riboprobe used for ribonuclease protection assays is also indicated at the bottom of the panel. B, the divergent amino-terminal amino acid sequences of our mouse TEF{alpha} and -{beta} are aligned with published mouse TEF{alpha} and rat TEF sequences. Carboxyl-terminal to residue 49 of mouse TEF{alpha} and residue 34 of TEF{beta}, the proteins have identical amino acid sequences. C, schematic representation of the mouse TEF gene. This structure was obtained by aligning cDNA sequences with the mouse genome using the BLAT alignment program (University of California Santa Cruz; www.genome.ucsc.edu). Black boxes represent exons and the thin line introns. The splicing pattern of TEF{alpha} and TEF{beta} is indicated.

 
TEF mRNA Is Widely Expressed in Adult Mouse Tissues and Cell Lines—To examine TEF mRNA distribution in mouse and rat tissues and cell lines, RNase protection analysis was utilized employing a probe corresponding to the 5' of TEF{beta}, allowing us to distinguish TEF{beta} (a 260-bp protected fragment) from TEF{alpha} (a 131-bp protected fragment) (Fig. 2). This analysis demonstrated that both TEF isoforms are expressed at high levels in mouse lung, bladder, kidney, brain, liver, and heart. However, the ratio of TEF isoforms varies in different tissues; for example, lung, kidney, and liver have similar expression of both isoforms, but TEF{alpha} is expressed at higher levels than TEF{beta} in bladder, uterus, ileum, heart, skeletal muscle, and placenta and in 10T1/2 fibroblasts as well as A10 vascular and intestinal visceral smooth muscle cells.



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  		FIG. 2.
RNase protection analysis of TEF expression in mouse and rat tissues and cells. A probe corresponding to the unique 5' of TEF{beta} was utilized to detect mRNA expression in different tissues and cells using RNase protection assay as described under "Materials and Methods." The riboprobe used protected a 260-bp fragment corresponding to mouse TEF{beta} and a 131-bp fragment corresponding to TEF{alpha}. The protected TEF{alpha} and -{beta} transcripts are indicated on the right of the panel. Riboprobes of known sizes as shown to the left of the panel were run alongside RPA samples to verify the size of the probe and protected fragments. GI SMC, intestinal smooth muscle cells. REF52, rat embryonic fibroblast cells.

 
TEF Binds to an AT-rich Region of the Telokin Promoter—To identify the TEF binding site on the telokin promoter, gel mobility shift assays were carried out using probes to either the AT-rich region and CArG box (AT/CArG), to the AT-rich region alone (AT), or to the CArG box alone (CArG) and purified TEF{alpha} protein. Two TEF·DNA complex bands were observed in assays using AT/CArG and AT probes, but not CArG probes. In contrast, SRF formed a mobility-shifted complex using the CArG probe, demonstrating the integrity of this probe. The two mobility-shifted complexes observed using purified TEF are consistent with previous data (14) and perhaps suggest that TEF can bind as a monomer or dimer (Fig. 3A). To verify the specificity of TEF{alpha} binding to the AT-rich region, competition experiments were performed using unlabeled oligonucleotides. A 50-fold excess of the unlabeled AT/CArG or AT oligonucleotides could compete for the binding to TEF{alpha} (Fig. 3A), whereas unlabeled CArG, mutant AT, E box, or AP2 could not compete for TEF{alpha} binding to the AT or AT/CArG probes. To further refine the TEF binding site within the AT-rich region, the ability of a series of AT/CArG mutant oligonucleotides containing single base pair mutations to compete for TEF or SRF binding to a wild type AT/CArG probe was determined. This analysis revealed that oligonucleotides containing mutations at positions A5, T6, A7, and T8 were unable to compete for TEF binding to the wild type probe, indicating that these residues are critical for TEF binding (Fig. 3B, upper panel). Mutations at T3, T4, A9, and A10 had decreased ability to compete for binding as compared with wild type probe, whereas all other mutations examined could compete for binding comparable to wild type probe. In contrast, all single base pair mutant oligonucleotides could effectively compete for SRF binding to the CArG box, and only the mutant with a deleted CArG box was unable to compete for SRF binding to the AT/CArG probe (Fig. 3B, lower panel). To further confirm the importance of this region for TEF{alpha} binding, AT/CArG probes containing single point mutations at two of these important residues, adenine at position 5 or 7, were selected for use as probes in gel mobility shift assays. As predicted, TEF was unable to form a DNA·protein complex with either of these mutant probes, although both probes still bound SRF (Fig. 3C). Together these data suggest that TEF directly binds to the sequence TTATATAA in the AT-rich region of the telokin promoter.



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  		FIG. 3.
Identification of the TEF binding sites on the telokin promoter. A, gel mobility shift assays were performed using probes containing either the AT-rich region and CArG box (AT-CArG), to the AT-rich region alone (AT), or to the CArG box alone (CArG) of the telokin promoter. Probes were incubated with 1 µg of purified TEF{alpha} in the presence of a 50-fold excess of cold competitor oligonucleotide as indicated, except in the far right hand lane (NONE*) in which purified serum-responsive factor (SRF) rather than TEF{alpha} was added. Following incubation for 15 min at room temperature, samples were run on a 4% polyacrylamide gel and mobility-shifted complexes visualized by autoradiography. AT MUT, AT region mutant probe; E box, telokin E box probe; AP2, a consensus AP2 binding site probe. B, to further refine the TEF binding site within the AT/CArG region, the ability of mutant AT/CArG oligonucleotides containing the indicated single base pair mutation to compete for TEF (upper panel) or SRF (lower panel) binding to a wild type AT/CArG probe was determined. C, to confirm the results from the competition studies described in panel B, probes were generated that correspond to two of these competitors and TEF binding to these mutant probes determined directly. AT/CArG probes containing mutations A5T or A7T or a wild type probe (WT) were used as probes for gel mobility shift assays. Probes were incubated with either purified TEF{alpha} or SRF as indicated. SRF produced a mobility-shifted complex with each of the probes, whereas TEF was only able to bind to the wild type probe.

 
TEF Specifically Activates Telokin Promoter Activity through Binding to AT-rich Region Element—To determine the function of TEF binding to the telokin promoter, we examined the ability of TEF to regulate a telokin promoter-reporter gene. The coding sequences of TEF{alpha} or TEF{beta} cDNAs were inserted into a mammalian expression vector and cotransfected into 10T1/2 or A10 cells together with a reporter plasmid consisting of a firefly luciferase gene under the transcriptional control of a 400-bp (–253 to +147) rabbit telokin proximal promoter region, which includes the AT-rich region and CArG box. Results from this analysis showed that TEF{alpha} and TEF{beta} increased telokin promoter activity 2.0- and 2.7-fold, respectively, in 10T1/2 fibroblast cells (Fig. 4A). In rat smooth muscle A10 cells, TEF{alpha} and -{beta} also stimulated promoter activity 1.2- and 1.4-fold, respectively (Fig. 4A). The lower levels of stimulation of the telokin promoter by TEF in A10 smooth muscle cells compared with 10T1/2 fibroblasts likely reflects the much higher basal levels of telokin promoter activity in smooth muscle cells as compared with fibroblasts.



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  		FIG. 4.
Effects of TEF{alpha} and -{beta} on telokin promoter activity. A, mouse TEF{alpha} and -{beta} or an empty expression vector plasmid were cotransfected together with a rabbit telokin promoter-luciferase construct (T400) and a thymidine kinase promoter-driven Renilla luciferase internal control plasmid into 10T1/2 fibroblasts or A10 smooth muscle cells as indicated. The level of promoter activity was determined by measurement of the firefly luciferase activity relative to the control Renilla luciferase. Promoter activity relative to vector control transfections is presented as mean ± S.E. of six samples. B, the ability of telokin promoters containing single base pair mutations equivalent to A5T or A7T in Fig. 3B to be activated by TEF{alpha} in 10T1/2 cells was determined as described in panel A. Values statistically different from controls are indicated by an asterisk (p < 0.001).

 
To directly correlate TEF binding and activation of the telokin promoter, mutant telokin promoters were generated that contained single base pair mutations that were shown to block TEF binding in gel mobility shift assays (Fig. 3B, A5T and A7T). As predicted, TEF was unable to activate these mutant telokin promoter reporter genes (Fig. 4B).

TEF Activation Is Promoter-specific—To determine whether TEF also activated the promoters of other smooth musclespecific genes, we examined its effects on reporter genes driven by the smooth muscle {alpha}-actin, SM22{alpha}, or the smooth muscle myosin heavy chain promoters. Results from these luciferase assays revealed that only the telokin promoter was significantly activated by TEF (Fig. 5).



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  		FIG. 5.
TEF activation is promoter-specific. TEF{beta} or empty expression vector plasmid was co-transfected together with the rabbit telokin promoter (T400), SM {alpha}-actin, SM22{alpha}, smooth muscle myosin heavy chain (SM-MHC), or minimal thymidine kinase promoter-luciferase reporter genes into 10T1/2 cells as indicated. Data presented are mean ± S.E. of 15 samples from three independent experiments. For each promoter, activity is normalized to a Renilla luciferase internal control and expressed relative to vector control transfections.

 
An Amino-terminal-truncated Form of TEF Acts as a Dominant Negative—To further evaluate the role of TEF in regulating telokin expression, a dominant negative TEF was generated by deleting the amino-terminal transcription activation domain (Fig. 1A). The TEF-DN was shown to directly bind to the AT-rich region of the telokin promoter in gel mobility shift assays and to compete with the wild type TEF{alpha} for DNA binding (Fig. 6A). In reporter gene assays the truncated TEF acted as a dominant negative and inhibited the ability of TEF{beta} to activate the telokin promoter in a dose-dependent manner (Fig. 6B). Consistent with results obtained using wild type TEF, the TEF-DN had no effect on the activity of the other smooth muscle-specific promoters analyzed (data not shown). These data suggest that the amino-terminal-truncated TEF acts as a dominant negative by competing with the wild type TEF for binding to the AT-rich region in the telokin promoter.



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  		FIG. 6.
Truncated TEF acts as a dominant negative. A, gel mobility shift assays were carried out using an AT-region/CArG probe from the telokin promoter as described under "Materials and Methods." Probes were incubated with 1 µg of purified TEF{alpha} or 1, 2, or 3 µl of E. coli extract containing His-tagged TEF-DN as indicated. Anti-His antibody was added to confirm the identity of the TEF-DN-containing complex (SS, supershifted complex). B, a telokin promoter-luciferase reporter gene was co-transfected together with either vector (Control), TEF{beta}, or TEF{beta} and increasing amounts of TEF-DN into 10T1/2 cells as indicated. Luciferase assays were performed as described under "Materials and Methods." Data presented are mean ± S.E. of six samples.

 
TEF Activation of the Telokin Promoter Is Calcium-dependent—Previous studies demonstrated that TEF activation of the inducible cAMP early repressor gene is increased by elevated intracellular calcium (22). Therefore, we examined the possibility that calcium regulates the TEF activation of the telokin gene. Thapsigargin, a drug that increases intracellular calcium by inhibiting endoplasmic reticular Ca2+-ATPase, was used to treat the 10T1/2 cells co-transfected with TEF and a telokin reporter gene. Results from these experiments revealed that thapsigargen enhances TEF activity up to 11-fold in a dose-dependent manner (Fig. 7).



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  		FIG. 7.
TEF activation of the telokin promoter is regulated by intracellular calcium. 10T1/2 cells were co-transfected with pcDNA HisA or pcDNAHis-TEF{beta} and a telokin promoter reporter gene (T400) and pRLTK internal control. 5 h after transfection, cells were treated with vehicle (DMSO, Me2SO) or 0.01 or 0.04 µM thapsigargin as indicated for an additional 18 h before harvesting and analysis of luciferase activity. Data presented are the mean ± S.E. of six samples. Activity, normalized to Renilla luciferase internal control, is expressed relative to vector control transfections.

 
TEF Up-regulates Endogenous Telokin Expression—To further examine the role of TEF in regulating telokin expression in vivo, the ability of adenoviral-expressed TEF to regulate endogenous smooth muscle protein expression in A10 smooth muscle cells was evaluated. A10 smooth muscle cells were infected with adenovirus encoding TEF{alpha}, TEF{beta}, TEF-DN, or {beta}-galactosidase. At 72 h after infection, cells were harvested and endogenous protein expression analyzed by Western blotting. This analysis revealed that in A10 cells TEF{alpha} and -{beta} overexpression induced telokin protein expression levels ~2.2- and 2.8-fold, respectively, as compared with cells infected with a control LAC adenovirus (Fig. 8, A and B). A significant 1.5- and 2-fold increase in SM22{alpha} and MLCK, respectively, was also observed following infection with TEF{beta} (Fig. 8A). However, TEF{alpha} overexpression decreased SM22{alpha} and MLCK expression 2-fold (Fig. 8B). None of the other proteins examined, including smooth muscle {alpha}-actin, calponin, or myosin light chain 20 showed any differences in expression. Conversely, overexpression of dominant negative TEF resulted in a 45% decrease in endogenous telokin protein expression, a 50% decrease in SM22{alpha}, and a 30% decrease in MLCK, respectively, without effecting the expression of the other proteins examined (Fig. 8C). Overexpression of TEF{alpha} or -{beta} in 10T1/2 cells was not able to induce expression of endogenous smooth muscle genes, suggesting that TEF alone is not sufficient to drive fibroblast cells to differentiate to smooth muscle cells (data not shown).



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  		FIG. 8.
Effects of TEF{alpha}, TEF{beta}, and TEF-DN overexpression on endogenous protein expression in A10 cells. A10 cells were seeded in 6-well plates overnight and then infected with adenovirus encoding LacZ, TEF, or TEF-DN as indicated in phosphate-buffered serum at a multiplicity of infection of 100 for 4 h at 37 °C. 72 h following infection, protein extracts were prepared from infected cells and analyzed by Western blotting. 15 µg of extract were analyzed in each lane. Each panel represents a single blot that was sequentially reacted with antibodies to each of the proteins indicated. When required, after each blot was reacted with an antibody, it was stripped and reprobed with second-step antibody to confirm that the primary antibody had been stripped from the blots before reacting with a subsequent antibody. The positions of molecular mass markers are indicated to the right of the blots. Graphs to the right of each blot represent the quantitation of expression levels (mean ± S.E.) of each protein from TEF-infected cells relative to LAC-infected cells. Proteins statistically significant from the control are designated by an asterisk (p >=0.05, Student's nonpaired t test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we identified two distinct forms of mouse TEF (TEF{alpha} and -{beta}) that can bind to the AT-rich region of the telokin promoter. TEF specifically activated telokin promoter reporter genes in 10T1/2 cells, and overexpression of TEF in A10 vascular smooth muscle cells resulted in increased expression of endogenous telokin in these cells.

TEF is a member of the PAR subfamily of bZIP proteins that includes albumin D box-binding protein (DBP), E2A-hepatic leukemia factor (23, 24), and VBP (the chicken homologue of TEF (21)). TEF was originally identified as a thyrotroph-restricted factor that is required to activate the thyroid stimulating hormone (TSH{beta}) gene (14). Although described as an anterior pituitary-restricted factor during embryonic development, TEF is more widely distributed in adult rats (25) and mice (Fig. 2), with high levels of expression observed in liver, kidney, brain, lung, and bladder. Because TEF isoforms are expressed in a variety of tissues, the activity of TEF alone cannot be sufficient for the activation of telokin expression or induction of the smooth muscle lineage. In support of this, adenoviral-mediated overexpression of TEF{beta} in 10T1/2 fibroblasts was not sufficient to induce telokin expression or expression of any other smooth muscle-specific proteins examined (Fig. 8 and data not shown). This would be consistent with a paradigm in which the phenotype of a smooth muscle cell is determined by the combined activity of a number of tissue-restricted and broadly expressed transcription factors.

Recent studies have suggested that TEF also plays important roles in regulating expression of other genes. For example, TEF has been shown to be involved in regulating the hematopoietic-specific promoter of the LMO2 gene (26). The PAR proteins display a high degree of sequence identity in their DNA-binding domains and in a conserved PAR region located aminoterminal of the basic DNA-binding domain (14). Consistent with their highly conserved DNA-binding domains, PAR proteins exhibit very similar DNA binding specificity in vitro and recognize the sequence RTTAYGTAAY (R, purines; Y, pyrimidines) (27, 28). Using a binding site selection assay, TEF was found to bind preferentially to the consensus sequence 5'-GTTACGTAAT-3', which is identical to the previously determined hepatic leukemia factor recognition site (29). Results from our gel mobility shift analysis suggest that the TEF binding site on the telokin promoter is TTATATAA (Fig. 3). This sequence is very similar to the core PAR consensus binding site, although missing flanking purine and pyrimidine residues at the 5'- and 3'-ends, respectively, and position 7 is an adenine instead of a guanine. These differences suggest that the binding of TEF to the telokin promoter is likely to be of lower affinity than its binding to a consensus binding site. A similar lower affinity TEF binding site has recently been reported in the inducible cAMP early repressor gene (22). In addition, it was shown that TEF activates inducible cAMP early repressor gene transcription via a Ca2+-dependent mechanism that involves CaMK IV. Consistent with these studies, our results also demonstrate that the activation of the telokin promoter by TEF is augmented by elevated intracellular calcium levels (Fig. 7). The calcium-dependent regulation of a smooth muscle-specific gene is particularly intriguing in light of the observations that stretch and contractility, two processes that lead to elevated intracellular calcium, are able to help establish and maintain the differentiated state of smooth muscle cells (30). These data suggest a model in which the beneficial effects of stretch and contractility on smooth muscle differentiation result, in part, from the calcium-dependent activation of transcription factors, such as TEF, that control the expression of smooth muscle differentiation genes.

The two isoforms of mouse TEF, TEF{alpha} and -{beta}, isolated in the current study are homologous to the previously described chicken VBP{alpha}/{alpha} and VBP{beta}/{alpha} isoforms, respectively (21). Alignment of the mouse TEF cDNA sequences with the mouse TEF gene and available expressed sequence tag cDNA sequences failed to provide any evidence of mouse TEF isoforms with alternatively expressed carboxyl termini similar to those described for VBP. The mouse gene structure is, however, consistent with the TEF{alpha} and -{beta} isoforms arising from alternative promoter and first exon usage as described from chicken VBP (Fig. 1). The ability of TEF{beta} to activate the telokin promoter and endogenous telokin expression appears to be greater than that of TEF{alpha} (Figs. 4 and 8), suggesting that the amino termini of the two isoforms play a role in regulating TEF function. This is further supported by the surprising opposing effects of the two isoforms on the regulation of the expression of endogenous SM22{alpha} and MLCK genes in A10 smooth muscle cells. TEF{beta} acts as a positive factor, increasing the expression of endogenous SM22{alpha} and MLCK proteins; in contrast, these proteins were down-regulated by TEF{alpha} overexpression. These changes in SM22{alpha} expression do not correlate with results from reporter gene assays in which we were not able to demonstrate any effect of either TEF isoform on the proximal 456-bp SM22{alpha} promoter (Fig. 5). A possible explanation for this discrepancy is that TEF may bind to a region of the SM22{alpha} gene outside of the –456 to +61-bp promoter used in our luciferase assays. The transcription activation domain of TEF has been shown to be able to directly bind to the RNA Polymerase II complex and recruit it to a promoter (31). The functional difference between TEF{alpha} and -{beta} may thus result from the unique amino termini modifying the ability of the adjacent transcription activation domain to interact with the RNA polymerase II complex. In support of this model, amino-terminal truncation of VBP was also shown to alter the activity of VBP in a cell-specific manner (21).

TEF, D box-binding protein, and hepatic leukemia factor have all been shown to display circadian expression patterns in mouse liver and kidney, with TEF and D box-binding protein being expressed at highest levels at 8 p.m. and at lowest levels at 8 a.m. (25, 32, 33). These observations suggest the possibility that perhaps downstream targets of TEF such as telokin may also be regulated in a circadian pattern. However, no circadian pattern of telokin protein expression was observed in gut and bladder tissues harvested at 8-h intervals during a 24-h period (data not shown). These findings may not be totally unexpected, because previous studies have demonstrated that the circadian pattern of expression of TEF and D box-binding protein is restricted to tissues such as liver and kidney (25).

The ability of TEF to activate telokin promoter reporter genes but not the other smooth muscle-specific reporter genes examined, together with the selective effects of TEF overexpression on endogenous telokin and SM22{alpha} genes, suggests that TEF regulates the activity of some but not all smooth muscle-specific genes. These data imply that in a single tissue distinct transcription factors may be involved in the regulation of expression of distinct groups of smooth muscle-specific genes.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL058571 and DK61130. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY540631 [GenBank] and AY540632 [GenBank] . Back

{ddagger} To whom correspondence should be addressed: Dept. of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5120. Tel.: 317-278-1785; Fax: 317-274-3318; E-mail: pherring{at}iupui.edu.

1 The abbreviations used are: MLCK, myosin light chain kinase; SRF, serum response factor; TEF, thyrotroph embryonic factor; TEF-DN, TEF dominant negative; SM, smooth muscle; MHC, myosin heavy chain; PAR, proline- and acidic-amino acid-rich; VBP, vitellogenin promoter-binding protein; LAC, {beta}-galactosidase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Gary Owens for the SM {alpha}-actin and SM-MHC luciferase reporter constructs and Gina Simon and Julia Azriel for expert technical assistance.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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