Transcriptional Regulation of the Prothrombin Gene in Muscle*

Sunghee KimDagger and Phillip G. Nelson

From the Laboratory of Developmental Neurobiology, NICHD, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Thrombin has been shown to mediate neurite retraction in neurons and synapse elimination at the neuromuscular junction. The presence of prothrombin mRNA has been demonstrated in brain and in muscle, but extra-hepatic regulation of the prothrombin gene has not been investigated. To identify cis-acting DNA elements involved in the expression of the prothrombin gene in muscle, we have isolated and analyzed a 1.3-kilobase pair promoter region of the mouse prothrombin gene. Using a series of transiently transfected plasmid constructs in which gene segments of the prothrombin promoter were linked to the luciferase gene, we have identified a sequence, -302 to -210, essential for prothrombin promoter activity in C2-myotubes. Fine analysis revealed that deletion of nucleotides between -248 and -235 eliminated prothrombin promoter activity in C2-myotubes. Furthermore, electrophoretic mobility shift assays demonstrated that a nuclear factor present in C2-myotubes, but not in C2-myoblasts or HepG2 hepatocytes, specifically binds to the sequence -241 to -225. Substitutional mutation of nucleotides -237 to -231 abolished myotube-specific promoter activity and inhibited the nuclear factor binding. Quantitative reverse transcription polymerase chain reaction demonstrated the expression of prothrombin mRNA in myotubes, but not in myoblasts, of primary, C2, and G8 muscle cells. This result correlates with the lack of prothrombin promoter activity in C2-myoblasts. The data thus suggest that a myotube-specific nuclear factor binds to a cis-acting sequence encompassing the core nucleotides -237 to -231 and plays a critical role in muscle-specific, differentiation-dependent expression of the mouse prothrombin gene.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Thrombin is synthesized and secreted from hepatocytes as a proteolytically incompetent zymogen form, prothrombin, the activation of which occurs at the sites of vascular injury. The function of thrombin has been extensively studied in the vascular system, and thrombin is known to play a key role in the maintenance of hemostasis (1). Recent reports have also identified extravascular cellular functions that are mediated by thrombin in the process of neural development. Thrombin has been shown to modulate the shape of astrocytes (2, 3) and to mediate neurite retraction in neuronal cells (4-7) and synapse elimination at the neuromuscular junction (8, 9). Most thrombin-mediated cellular effects are observed experimentally with the addition of exogenous thrombin or thrombin inhibitors to an in vitro culture system. The cellular localization of non-hepatic prothrombin mRNA is of interest because most thrombin-mediated cellular effects observed in vitro are thought to occur in the normal course of development, such as synapse reshaping. The recruitment of activated thrombin from blood in the absence of vascular injury during these events is unlikely. In support of this scenario, the expression of prothrombin mRNA has been demonstrated in brain and neural cell lines (10, 11), as well as in rodent skeletal muscle and primary skeletal muscle cultures (9, 12). Moreover, thrombin proteolytic activity was detected in the supernatants of mouse muscle cultures (12), and in extracts of mouse skeletal muscle (9). However, the transcriptional regulation of prothrombin gene expression in tissues other than the liver has not been studied.

We have investigated the expression of the mouse prothrombin gene in muscle, and in this report, we demonstrate that transcriptional regulation of the mouse prothrombin gene in skeletal muscle cells is distinct from that in liver cells. Furthermore, the prothrombin gene is not expressed in myoblasts but is activated upon differentiation of skeletal muscle in culture.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Isolation of the Mouse Prothrombin Promoter Region-- The 5'-flanking region of the mouse prothrombin gene was cloned using the Genome Walker Kit (CLONTECH). The kit provides five sets of mouse genomic libraries generated from mouse genomic DNA cut with different restriction enzymes and ligated to short adapter sequences. Primary and nested polymerase chain reactions (PCRs)1 were performed using forward primers complementary to the adapter sequence and reverse primers, 5'-TACCAGGCTAACCAGGGCAGCCAGAGC-3' and 5'-AGCCAGAGCCAAGCAGCCAGGGGAGGCC-3', which correspond to nucleotides 84-58 and nucleotides 66-37, respectively, of the mouse prothrombin cDNA sequence (13). A 1.3-kilobase pair (kbp) PCR product was cloned into the pCR 2.1 vector (Invitrogen), designated as pCR/1.3-kbp, and sequenced on both strands by a commercial service (Lark Technologies, Inc.).

To determine the transcription start site of the prothrombin gene, poly(A)+ mRNA from the leg muscles of embryonic day 16 mice was isolated using the Oligotex Direct mRNA purification kit (Qiagen). Muscle mRNA (100 ng) was subjected to the 5'-RACE, Version 2.0 (Life Technologies, Inc.). Reverse primers 5'-CACTGAATCACACACACTGTGT-3' and 5'-TCCTCCAGGAAGCCACTGTT-3', corresponding to nucleotides 300-281 and 170-151, respectively, of the mouse prothrombin cDNA sequence, were employed in primary and nested PCRs (13). Putative clones were identified by EcoRI and ApaI/SacII restriction enzyme digestion analysis, and four clones were sequenced using the T7 Sequenase kit (Amersham Pharmacia Biotech). All animals used for the study were handled in accordance with the National Institutes of Health Animal Care and Welfare protocol.

Reporter Plasmids-- Prothrombin promoter gene segments containing various 5'-ends were generated by PCR using the pCR/1.3-kbp plasmid as a template. Forward primers used for PCR are shown in Table I. Promoter sequences containing substituted nucleotides, sequences M1 to M5, were also generated by PCR using forward primers with the indicated nucleotide substitutions (Table I). The numbering of nucleotides is relative to the transcription start site (+1) determined from the 5'-rapid amplification of cDNA ends. A common reverse primer, 5'-GTGAGATCTAGTGTGTCAGCTCCTG-3', contained a BglII site to facilitate cloning into the pGL3-basic vector (Promega) linearized with SmaI/BgllI. PCR was performed utilizing pfu polymerase (Stratagene). All the reporter plasmid constructs were sequenced on both strands to confirm their fidelity to the sequence of the 1.3-kbp mouse prothrombin gene. A series of pure clones was isolated by two consecutive platings on ampicillin-containing LB agar plates. Overnight cultures of 300 ml were subjected to the Maxi DNA preparation kit (Qiagen), and approximately 400 µg of plasmid was obtained. The stock reporter plasmids were diluted to 2 mg/ml in a buffer containing 10 mM Tris-Cl and 1 mM EDTA (TE), and kept at 4 °C.

                              
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Table I
Forward primers used for the construction of reporter plasmids

Cell Culture, Transfection, and Luciferase Assay-- The mouse C2 skeletal muscle cell line was obtained from Dr. Christian Fuhrer (National Institutes of Health, Bethesda, MD) and maintained at 37 °C in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum. The HepG2 liver cell line was purchased from ATCC (Rockville Pike, MD) and maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Transient transfection was performed by the method of calcium phosphate/DNA co-precipitation using a commercial kit (Life Technologies, Inc.). For the transfection of C2-myoblasts, mononucleated C2 cells at approximately 40% confluency were transfected, and the cells were harvested 48 h later. No signs of fusion were observed microscopically at the time of harvesting. For C2-myotubes, C2 cells 80-90% confluent were transfected with reporter constructs. After 24 h, the medium was replaced by Dulbecco's modified Eagle's medium supplemented with the 5% horse serum to induce differentiation into multinucleated myotubes. Within 48 h, fusion was complete, and C2-myotubes were harvested for the luciferase assay. pRL-CMV vectors were co-transfected with pGL3 reporter plasmids to normalize the pGL3 reporter luciferase activity according to the dual-luciferase reporter assay system (Promega). Measurements were made using a Lumat LB 9507 luminometer (EG & G Berthold, Wilbad, Germany).

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear protein extracts from C2-myoblasts, C2-myotubes, and HepG2 cells were prepared as described (14) with the modifications described in Ref. 34. Nuclear extracts were dialyzed in a buffer containing 20 mM HEPES (pH 7.9), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, and 5 mM dithiothreitol at 4 °C. Any precipitation formed during dialysis was removed by centrifugation. Protein concentrations were determined using a commercially available Bradford assay (Bio-Rad). For the EMSA probes, wild type (WT) and five mutant constructs (M1-M5) containing nucleotides from -284 to -140 of the prothrombin promoter sequence were generated by PCR amplification using the pCR/1.3-kbp construct as a template. PCR was performed with WT or mutant forward primers (Table I) and a common reverse primer, 5'-AGGTTCTCCTGGAACCAG-3' (-140 to -157). PCR products were purified by low melting point agarose gel electrophoresis and end-labeled with [gamma -32P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech) using T4 polynucleotide kinase (Life Technologies, Inc.) and separated from free nucleotides by Sephadex G-25 chromatography. Oligonucleotides matching short segments of the WT fragment were synthesized by a commercial service (Genosys) and purified by denaturing polyacrylamide gel electrophoresis. For competitive EMSA, the double-stranded oligomeric probes were prepared by annealing equal amounts of complementary oligonucleotides. A 100-fold excess of unlabeled probe was added in competitive binding reactions. DNA-protein binding reactions were carried out in a total volume of 15 µl, which included 2 µg of nuclear proteins, 1 µg of poly(dI)·poly(dC), 50 fmol of probes (~20,000 cpm), and a buffer consisting of 5% glycerol, 0.5 mM EDTA, 50 mM NaCl, 10 mM Tris-Cl, pH 7.5. After incubation at room temperature for 30 min, DNA-protein complexes were separated from the free oligonucleotides by electrophoresis on a 4% polyacrylamide gel in buffer containing 50 mM Tris base, 380 mM glycine, and 2 mM EDTA. The gel was dried and autoradiographed.

Quantitative Reverse Transcription-PCR-- RNA samples from primary muscle and fibroblast cultures and skeletal muscle cell lines C2 and G8 were isolated using TRIzol (Life Technologies, Inc.). Primary mouse muscle cultures were prepared as described in Ref. 12, and primary fibroblasts were obtained from the preplating used to remove contaminating fibroblasts during muscle culture preparation. The G8 cell line was obtained from ATCC and maintained in Dulbecco's modified Eagle's medium with 10% horse serum and 10% fetal calf serum. G8 cells differentiated into multinucleated myotubes when at confluency without a reduction in serum content of the medium. cDNA was synthesized from 1 µg of total RNA by priming with oligo(dT)18 using the Advantage RT-for-PCR kit (CLONTECH).

Competitor mimic cDNAs flanked by the target gene primer sequences (5'-CAACATCAATGAGATACAGCCCAGCGTCC-3' and 5'-CATGCGTGTAGAAGCCGTATTTCCCCTTC-3' for prothrombin and 5'-GGCCAGATCCTGATGCCCAAC-3' and 5'-CAGCTGTGCTGCTCTTTCTAC-3' for rPL32) were generated using a PCR mimic construction kit (CLONTECH). The mimic cDNAs were initially diluted 10-fold ranging from 100 attomoles/µl to 10-6 attomoles/µl and co-amplified with a constant amount of the muscle cDNA sample in a 25-µl reaction that included 1× PCR buffer, 50 µM dNTPs, 0.1 µM each 5'- and 3'-primers, and 1.2 units of Taq polymerase (Perkin-Elmer). Samples were amplified for 30 cycles at 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 1 min, and the PCR products were subjected to electrophoresis on 4-20% gradient polyacrylamide gels (Novex). After the initial screening using 10-fold serially diluted competitor cDNA, the optimal range of mimic cDNA dilution was chosen for the competitive PCR experiment described in Fig. 1. The intensities of bands in EtBr-stained gels were quantified using the BioImage IQ program (BioImage, Ann Arbor, MI) and used to calculate the ratios of prothrombin to mimic cDNA. The values were then normalized relative to the values obtained for a housekeeping gene, the gene encoding ribosomal protein L32 (rPL32) (15).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Characterization of the Promoter Region of the Mouse Prothrombin Gene-- The DNA sequence of the mouse prothrombin 1.3-kbp clone has been submitted to GenBankTM. The sequence between -972 and the translation initiation codon ATG had 69% identity with the corresponding region of the human prothrombin gene, based on a GCG sequence alignment program (Genetics Computer Group, Inc., Madison, WI). The human prothrombin sequence used for the alignment was as previously reported (16) with GenBankTM accession number M65141. The sequence between -1211 and -1017 was 86% identical to the mouse B1 repetitive sequence (17) found in numerous mouse genes based on a National Center for Biotechnology Information BLAST search (18). The transcription start site was determined to be five nucleotides upstream from the translational initiation codon ATG based on sequence analyses of the four clones resulting from the 5'-rapid amplification of cDNA ends. The promoter region of the mouse prothrombin gene does not contain a consensus CCAAT motif or a canonical TATA box, as has been reported for the human prothrombin gene (16). However, we have located a TATA-like sequence, TATTAA, at -46 with respect to the transcription start site.

Myotube-specific Expression of Prothrombin mRNA-- mRNA expression of mouse prothrombin was investigated in primary mouse muscle cultures and in the mouse skeletal cell lines C2 and G8 (Fig. 1). A competitive PCR was used to quantitatively determine differences in prothrombin mRNA expression. The expression of prothrombin mRNA could be detected in myotubes of primary, C2, and G8 cells. Prothrombin mRNA in mononucleated myoblasts both from primary and from C2 and G8 was undetectable. The quantitation results of the competitive PCR on rPL32 demonstrated that housekeeping rPL32 mRNA levels were equivalent in myoblasts and myotubes (Fig. 1), thus confirming that prothrombin mRNA is present in differentiated multinucleated myotubes but not in undifferentiated myoblasts of primary, C2, and G8 cells. The expression of prothrombin mRNA was also not detected in the contaminating primary fibroblasts obtained during the muscle culture preparation (Fig. 1).


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Fig. 1.   Competitive PCR showing the myotube-specific expression of mouse prothrombin mRNA. The competitor cDNAs for prothrombin (10-3 amol) and for rPL32 (1 amol) were co-amplified with equal amounts of various cDNA samples. Samples were amplified for 30 cycles at 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 1 min, and the PCR products were subjected to electrophoresis in 4-20% gradient polyacrylamide gels and stained with EtBr. The intensity of bands was quantified, and the ratio of prothrombin to competitor cDNA was calculated. The ratio was then normalized relative to the ratio of the housekeeping gene, the gene encoding rPL32. The + and - signs indicate the myotube stage (multinucleated) and myoblast stage (mononucleated), respectively. M represents a 100-bp DNA marker (Life Technologies, Inc.).

Differential Activity of the Mouse Prothrombin Promoter in C2-Myotubes and HepG2 Cells-- To investigate the contributions of specific promoter regions to prothrombin gene expression in muscle and liver, varying lengths of the mouse prothrombin gene promoter possessing the same 3'-end sequence were linked to the 5'-end of a promoterless luciferase reporter gene in the pGL3-basic plasmid vector. The reporter plasmid constructs were initially tested in human HepG2 hepatocytes to investigate the promoter activity of mouse prothrombin constructs in relation to that previously reported for the human prothrombin promoter in HepG2 cells (16). The overall pattern of activity of the mouse reporter constructs in HepG2 cells (Fig. 2) was nearly identical to that of the human prothrombin reporter constructs tested in HepG2 cells (16). In the human gene, a liver-specific cis-acting sequence was shown to be located between -940 and -860 (for numbering of the human gene, see Ref. 16), and deletion of nucleotides in this region virtually destroyed the activity of the human prothrombin gene promoter in HepG2 cells (16). The GCG best-fit alignment indicated that residues -940 to -840 in human prothrombin align with -816 to -723 in the mouse prothrombin sequence (data not shown), and deletion of this region in mouse also drastically diminished promoter activity in HepG2 cells (Fig. 2). In addition, no significant differential activity was observed among mouse promoter segments terminating between nucleotides -607 and -77 (Fig. 2), similar to the case of human gene (16). As described for the corresponding sequence of human prothrombin, the mouse sequence -816 and -723 was also flanked by Sp-1-like sequences. Moreover, a putative liver-specific enhancer AATATTT (hepatic nuclear factor 1) identified in the human promoter is also conserved in the mouse prothrombin gene.


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Fig. 2.   Deletion analysis of the promoter region of the mouse prothrombin gene. C2-myoblasts, C2-myotubes, and HepG2 cells were transiently transfected with an expression vector containing the luciferase gene driven by various 5'-end deletion of the mouse prothrombin promoter. For C2-myotubes, the transfections were performed in the myoblast stage, and fusion was completed within 72 days of transfection in the presence of 5% horse serum. Values reported represent activity relative to the minimal promoter construct (-77/+5). Data are the mean ± S.E. of three to five independent experiments using at least triplicate samples. The analysis reveals that the construct -802/+5 exhibits the highest promoter activity and that the proximal sequence between -302 and -210 is critical for promoter activity of the prothrombin gene in C2-myotubes. The transfections were carried out at least in triplicate with n greater than 3. The final results are expressed as means ± S.E. of values relative to the minimal size construct -77/+5.

The above constructs were also transiently transfected into C2 mouse muscle cells to investigate the muscle-specific regulation of mouse prothrombin expression (Fig. 2). None of the constructs had appreciable luciferase activity in C2-myoblasts (Fig. 2). This result correlates well with the lack of expression of the endogenous prothrombin gene in undifferentiated C2-myoblasts (Fig. 2). Highest luciferase activity in C2-myotubes was from the -802/+5 construct (29-fold above the construct -77/+5). The constructs -1218/+5 and -984/+5 showed 2-fold lower activity in C2-myotubes compared with that of -802/+5, whereas the activities of all three constructs were equivalent in HepG2 cells (Fig. 2). These results suggest that the region -984 to -802 encompasses a negative cis-element that represses expression of the mouse prothrombin in C2-myotubes but not in HepG2 cells. Moreover, the constructs terminating between -607 and -210 did not show dramatically lower activity in C2-myotubes as they did in HepG2 cells (Fig. 2). The proximal promoter region -302/+5 still possessed a 17-fold higher activity than that of -77/+5 and was about 60% as active as the -802/+5 construct. These results indicate that the transcriptional regulation of prothrombin gene in C2-myotubes is distinct from that in HepG2 cells.

Localization of a cis-Acting Element in the Proximal Promoter region of the Mouse Prothrombin Gene-- Five additional reporter plasmids were constructed by progressive deletion of 5' sequence spanning -302 to -210 and were transfected into C2-myotubes (Fig. 3). The luciferase activities of the reporter constructs are shown relative to the activity of the construct -302/+5 as 100% activity. The -235/+5 construct was about half as active as the longer constructs. Therefore, the proximal sequence between nucleotides -248 to -235 may contain an important cis-acting regulatory sequence that is required for prothrombin promoter activity in C2-myotubes.


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Fig. 3.   Deletion analysis of the proximal promoter sequence -302 to -210 of the mouse prothrombin gene. C2-myotubes were transiently co-transfected with pGL3 prothrombin reporter vectors and the pRL-CMV control vectors. Values are normalized relative to values for the co-transfected CMV vector, and the luciferase activity is shown relative to the activity of the construct 302/+5, taken as 100%. Data are mean ± S.E. of four independent experiments using triplicate samples. The analysis indicates that a cis-acting sequence essential for promoter activity in C2-myotubes is located in the sequence containing nucleotides -248 and -235.

Detection of trans-Acting Factors in C2-Myotube Nuclear Extracts-- In pursuit of finding prothrombin gene-specific trans-acting factors in C2 nuclear extract, EMSA was performed with the probe -248/-140 and the nuclear extracts of C2-myoblasts, C2-myotubes, and HepG2 cells (Fig. 4). The results demonstrated that complex C-I was present only in the nuclear extracts from C2-myotubes. Other complexes (C-II, C-III, and C-IV) were shared between C2-myoblast and C2-myotube nuclear extracts. The complexes CB-I and CB-II were observed either exclusively or noticeably higher in C2-myoblasts nuclear extracts. There was no apparent binding of nuclear factors from HepG2 cells specifically in the area of the C-I complex.


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Fig. 4.   A nuclear factor present in C2-myotubes binds to the probe -248/-140. Nuclear extracts from C2-myotubes, C2-myoblasts, and HepG2 cells were used in EMSA with the probe -48/-140. Nuclear extracts (2 µg) were incubated with the 32P-labeled probe (~20,000 cpm) for 30 min at room temperature. DNA-protein complexes were separated by electrophoresis on 4% polyacrylamide gels. Complex C-I is present only in C2-myotubes, not in C2-myoblasts or in HepG2 cells. C2-myoblasts nuclear extracts from relatively light (i) and heavy (ii) cultures were also compared. C, complex; CB, complex formed with myoblasts nuclear extracts.

The competitive EMSA demonstrated that the binding of both C-I and C-II to -248/-140 could be inhibited in the presence of excess cold probe -241/-210 or -241/-225 (Fig. 5). The cold probe -209/-166 preferentially inhibited complex C-III binding to the hot probe -248/-140 and also appeared to affect the binding of C-II. However, this indirect inhibition of the binding of C-II was not observed when we performed a competitive EMSA identical to that described in Fig. 5 using C2-myoblasts nuclear extracts (data not shown). Interestingly, we found that the formation of the both complexes C-II and CB-I was inhibited in the presence of either cold competitors, -241/-210 or -241/-225, as was the case for myotube complexes C-I and C-II. These results suggested that the C-II protein may play a central role in the binding of either the myoblast complex CB-1 or the myotube complex C-I during differentiation of C2 cells.


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Fig. 5.   Competition of the unique C2-myotube transcriptional factor binding to the probe -248/-140 with short oligomeric probes. Nuclear extracts from C2-myotubes were used in a competitive EMSA with the probe -48/-140. C2 myotube nuclear extracts (2 µg) were incubated with the 32P-labeled probe (~20,000 cpm) in the presence of a 100-fold excess of unlabeled probes as indicated at the top of the figure. After incubation for 30 min at room temperature, DNA-protein complexes were separated by electrophoresis on 4% polyacrylamide gels. The specific binding of C-I to the probe -248/-140 was selectively inhibited in the presence of the competitor probes -241/-210 and -241/-225.

Mutational Analysis of the cis-Acting Sequence-- Because the oligomeric probe -241/-225 successfully inhibited the binding of C-I nuclear factor to the WT probe -248/-140 (Fig. 5), the region -241 to -227 was mutated by step-wise substitution of adenine nucleotides (Fig. 6A). A nearly complete inhibition of the binding of complex C-I was observed with the mutant probes M2 and M3. The results were in agreement with transfection analysis of the five mutant reporter plasmid constructs (Fig. 6B). The luciferase activity of the constructs was evaluated relative to the activity of the WT construct. Constructs containing mutations M2 (-237/-235) and M3 (-233/-231) had diminished activity, comparable to the activity loss observed with the -235/+5 construct. The M1 mutation resulted in a moderate inhibition of the C-I binding but did not result in a significant decrease in reporter activity. Taken together, these results indicate that C-I interacts with the cis-region encompassing the core nucleotides -237 to -231 and plays a critical role in prothrombin reporter activity in C2-myotubes.


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Fig. 6.   Substitution of the nucleotides between -237 and -231 inhibits the binding of C-I (A) and destroys the prothrombin promoter activity in C2-myotubes (B). A, the mutant probes were generated by PCR using primers with adenine nucleotide substitutions as indicated. The 32P-labeled WT and mutant probes M1-M5 (~20,000 cpm) were incubated with C2-myotube nuclear extracts (2 µg) for 30 min at room temperature. DNA-protein complexes were separated by electrophoresis on 4% polyacrylamide gels. The TTGATTC oligonucleotides (-237/-231) were shown to be essential for the binding of C-I. B, C2-myotubes were transiently transfected with WT (-248/+5) or mutant (M1-M5) reporter plasmids. Values are normalized relative to values for the cotransfected pRL-CMV vector, and the luciferase activity is shown relative to the activity of the WT construct taken as 100%. Data are mean ± S.E. of three independent experiments using triplicate samples. The analysis indicates that a cis-acting sequence crucial for the prothrombin promoter activity in C2-myotubes is located in nucleotides -237 to -231.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Until the early 1990s, prothrombin was considered to be a model gene for studying liver-specific gene regulation because of its abundant and exclusive expression in the liver (16). Recent reports, however, strongly indicate that the regulation of prothrombin gene expression in the skeletal muscle might be independent of that in the liver. In particular, the expression of prothrombin mRNA in muscle appears to be innervation-dependent, being high at birth and drastically diminishing after the period of stable synapse formation (9). Moreover, we have recently found that the denervation of adult limb muscle significantly up-regulates the expression of prothrombin.2 Nevertheless, the localization of prothrombin mRNA in muscle has been problematic due to extremely low mRNA levels. In our experience, the expression of prothrombin mRNA in neonatal mouse skeletal muscle was up to 3 orders of magnitude lower than in neonatal and adult mouse liver. Although a sensitive reverse transcription-PCR analysis detected the expression of prothrombin mRNA in the leg muscle at mouse embryonic days 12 and 16 (9), the cellular localization of prothrombin using in situ hybridization revealed the expression of prothrombin exclusively in the liver of the corresponding days of embryos (19). It has been reported recently that application of exogenous thrombin to C2-myoblasts resulted in a delay in the formation of myotubes without being mitogenic (20). However, in that study, the authors did not investigate endogenous prothrombin expression in C2 cells. Earlier, exogenous thrombin had been shown to be mitogenic in rat primary myoblasts (21). Using a quantitative reverse transcription-PCR, we have demonstrated here that prothrombin mRNA is exclusively expressed in differentiated myotubes but not in myoblasts of both primary muscle culture and skeletal muscle cells C2 and G8. The prothrombin message appears upon fusion of muscle cells, and thus, it is intriguing to speculate about the in vivo function of endogenous prothrombin in the muscle fibers. The proteolytic activity of thrombin has already seen associated with the process of synapse elimination at the neuromuscular junction (8, 9).

Our experiments revealed that the activity of the prothrombin gene in C2-myotubes was significantly dependent on a C2-myotube nuclear factor C-I binding to the sequence between -241 and -225 encompassing the nucleotides TGATTCA (-236/-230), a sequence previously reported to bind to AP-1 (22). The substitution of either nucleotides -237/-235 or -233/-231 abolished both the binding of C-I and reporter activity in C2-myotubes. A major component of the AP-1 binding factor, c-Jun, is known to form homodimers as well as heterodimers with Jun B, Jun D, c-Fos, Fra-1, Fra-2, and Fos B. A putative AP-1 binding site is located in a corresponding region of the human prothrombin sequence based on a search using Transcription Element Search Software on the World Wide Web.3 We therefore performed both supershift EMSA and EMSA coupled with Western blot analysis (data not shown) using antibodies (purchased from Santa Cruz Biotechnology) specific to c-Jun (cross-reactivity with c-Jun, Jun B, and Jun D) and c-Fos (cross-reactivity with c-Fos, Fos B, Fra-1, and Fra-2). In addition, we have performed competitive EMSA with the 26-mer DNA probe containing a consensus AP-1 binding motif TGACTCA (Santa Cruz Biotechnology). None of the antibodies alone or combined showed a reactivity with the C2-myotube nuclear complex C-I. The 26-mer AP-1 sequence failed to compete for the binding of C-I to the -248/-140 probe (data not shown), whereas the 16-mer oligomeric -241/-225 probe, encompassing the sequence TGATTCA, competitively abolished the binding of C-I to -248/-140. It has been previously reported that the expression of c-jun mRNA is down-regulated during differentiation of C2 cells and that the constitutive expression of c-Jun in C2-myoblasts inhibits the myogenic differentiation of C2 cells (23). Thus, we have concluded that the possibility of the C-I complex containing AP-1 is remote. We are currently engaged in purification of the C-I factor, and further analysis will determine its identity.

A family of myogenic basic helix-loop-helix DNA-binding proteins, mainly MyoD, Myf5, myogenin, and MEF4, play a pivotal role in the determination of muscle lineage and the differentiation of skeletal muscle fibers (reviewed in Refs. 24 and 25). These myogenic factors bind E-box motifs (CANNTG) found in control regions of muscle-specific genes, and the binding of the myogenic factors to the E-box has been shown to play a critical role in the expression of muscle-specific genes (reviewed in Refs. 24 and 25). We have identified four E-box motifs in the 1.3-kbp promoter region of the mouse prothrombin gene. However, the three E-boxes located between -1218 and -802 did not play a positive role in reporter activity in transient transfection because the highest luciferase activity was obtained with the construct -802/+5. Also, the construct -151/+5, possessing a proximal E-box, did not exhibit any appreciable level of luciferase activity in C2-myotubes (Fig. 2). Although maximal expression of many muscle-specific genes is controlled by the presence of more than one E-box, it has been reported that certain muscle-specific genes can be controlled by a single E-box accompanied by other regulatory sites (26-28). There have also been examples of muscle-specific genes that do not contain E-boxes within their regulatory regions (29-31). Such regulatory regions were shown to contain motifs, such as CArG (29), M-CAT (30), and MEF-2 (32, 33), that were also shown to participate in the activation of muscle-specific genes. However, the C-I binding site, CTTATTGATTC (-241/-231), does not resemble CArG, M-CAT, or MEF-2 sequences. Whether myogenic factors interact in cis with the C-I factor or C-I is indirectly activated by myogenic factors during muscle differentiation remains to be determined.

The myotube-specific complex C-I readily interacts with the probe -248/-140; however, C-I did not bind to the oligomeric probes -241/-210 and -241/-225. Instead, a ubiquitous nuclear factor (not AP-1) present in both C2-myoblasts and C2-myotubes bound to these two probes, as did nuclear factors in HepG2 cells (data not shown). Most importantly, there was no evidence of differential binding activity of C2-myotube nuclear extracts to either -241/-210 or -241/-225 probes in comparison with nuclear extracts from C2-myoblasts and HepG2 cells. Nevertheless, when these probes were used in excess in the competitive EMSA, they could compete the binding of C-I to the probe -280/-140. This might indicate a cooperative interaction between the C-I complex and nuclear complexes bound to the -209 to -148 that resulted in an increased binding affinity of C-I to its site.

In this report, we have demonstrated that regulation of the prothrombin gene in muscle is quite distinct from that in liver and that its activation is dependent upon the terminal differentiation of muscle cells. Detection of the endogenous prothrombin mRNA in C2-myotubes but not in myoblasts is in agreement with the observation that the prothrombin promoter is active in C2-myotubes but not in C2-myoblasts. It has been shown that exogenously applied thrombin inhibitors can completely block activity-dependent synapse elimination in a nerve-muscle co-culture system (8) and that cholinergic stimulation of aneural myotubes results in the augmentation of prothrombin mRNA expression (12). It will thus be interesting to investigate whether the cis-acting sequence TTGATTC (-237 to -231), identified here to be critical for the transcription in differentiated muscle, can also confer activity-dependent regulation of the prothrombin gene in myotubes.

    ACKNOWLEDGEMENTS

We express our appreciation to Drs. Gretchen Gibney and James Brady for their critical reading of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The 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/EMBL Data Bank with accession number(s) AF049131.

Dagger To whom correspondence should be addressed: Bldg. 49, Rm. 5A38, Laboratory of Developmental Neurobiology, NICHD, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-402-1484; Fax: 301-496-9939; E-mail: shkim{at}helix.nih.gov.

1 The abbreviations used are: PCR, polymerase chain reaction; kbp, kilobase pair(s); EMSA, electrophoretic mobility shift assay; WT, wild type.

2 S. Kim, unpublished data.

3 J. Schug and G. C. Overton (1997) Technical report CBIL-TR-1997-1001-v0.0, Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania, (available at agave.humgen.upenn.edu/tessl).

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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