Cardiac myotrophin exhibits rel/NF-kappa B interacting activity in vitro.

Myotrophin is a soluble-12 kilodalton protein isolated from hypertrophied spontaneously hypertensive rat and dilated cardiomyopathic human hearts. We have recently cloned the gene coding for myotrophin and expressed it in Escherichia coli. In the present study, the expression of myotrophin gene was analyzed, and at least seven transcripts have been detected in rat heart and in other tissues. We have further analyzed the primary structure of myotrophin protein and identified significant new structural and functional domains. Our analysis revealed that one of the ankyrin repeats of myotrophin is highly homologous specifically to those of IκBα/rel ankyrin repeats. In addition, putative consensus phosphorylation sites for protein kinase C and casein kinase II, which were observed in IκBα proteins, were identified in myotrophin. To verify the significance of these homologies, κB gel shift assays were performed with Jurkat T cell nuclear extract proteins and the recombinant myotrophin. Results of these assays indicate that the recombinant myotrophin has the ability to interact with NF-κB/rel proteins as revealed by the formation of ternary protein-DNA complexes. While myotrophin-specific antibodies inhibited the formation of these complexes, rel-specific p50 and p65 antibodies supershifted these complexes. Thus, these results clearly indicate that the myotrophin protein to be a unique rel/NF-κB interacting protein.

Myotrophin is a soluble-12 kilodalton protein isolated from hypertrophied spontaneously hypertensive rat and dilated cardiomyopathic human hearts. We have recently cloned the gene coding for myotrophin and expressed it in Escherichia coli. In the present study, the expression of myotrophin gene was analyzed, and at least seven transcripts have been detected in rat heart and in other tissues. We have further analyzed the primary structure of myotrophin protein and identified significant new structural and functional domains. Our analysis revealed that one of the ankyrin repeats of myotrophin is highly homologous specifically to those of IB␣/rel ankyrin repeats. In addition, putative consensus phosphorylation sites for protein kinase C and casein kinase II, which were observed in IB␣ proteins, were identified in myotrophin. To verify the significance of these homologies, B gel shift assays were performed with Jurkat T cell nuclear extract proteins and the recombinant myotrophin. Results of these assays indicate that the recombinant myotrophin has the ability to interact with NF-B/rel proteins as revealed by the formation of ternary protein-DNA complexes. While myotrophin-specific antibodies inhibited the formation of these complexes, rel-specific p50 and p65 antibodies supershifted these complexes. Thus, these results clearly indicate that the myotrophin protein to be a unique rel/NF-B interacting protein.
Cardiac myocyte cell hypertrophy has been used as an in vitro model for studying cardiac hypertrophy. Cardiac myocytes respond to hemodynamic overload by altering the expression of specific set of genes, which are needed for hypertrophy. Our laboratory has been studying the molecular basis of myocardial hypertrophy using spontaneously hypertensive rat as an animal model (1)(2)(3). Earlier, Sen et al. (1,2) isolated a novel 12-kilodalton protein, which we named myotrophin, from the hypertrophied ventricles of spontaneously hypertensive rat (1) and dilated cardiomyopathic human hearts (2) based on its ability to stimulate protein synthesis specifically in cardiac myocytes (1). Recently, we have isolated the cDNA clones encoding rat myotrophin (4) 1 and found that the cardiac myotrophin is identical to a previously reported rat brain v1 protein (7, 8) whose function is not determined at present in brain. In addition, we have expressed the myotrophin protein in Escherichia coli and showed that the recombinant myotrophin has the ability to stimulate protein synthesis in neonatal cardiac myocytes (4). 1 In the present study, analyzed the expression of myotrophin in various tissues, identified new structural and functional domains, and, using the recombinant myotrophin, determined one of the key activities of myotrophin.

EXPERIMENTAL PROCEDURES
Northern Analysis of Myotrophin mRNAs-RNA transcripts specific for myotrophin were analyzed in various rat tissues. Total RNA was first isolated from 9-day-old rat hearts. Poly(A)-enriched RNA was isolated using the oligo(dT) method. Briefly, total RNA was applied to an oligo(dT)-cellulose (Collaborative Research type III) column at high salt conditions (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 M NaCl). After washing the column with high-salt buffer, poly(A) RNA was eluted with no-salt (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) buffer. The poly(A) RNA eluted by this method was fractionated on 1% agarose formaldehyde gels, transferred to a ZetaProbe membrane, and hybridized with radiolabeled probe. Random-Primer labeling method was used to generate radiolabeled myotrophin cDNA probe using pCRII-8-Myo cDNA clone (4). 1 Utilizing the same clone, single-stranded myotrophin-specific radiolabeled RNA probe was made using T7 RNA polymerase-directed in vitro transcription system. The hybridization experiment was done using very high stringency and wash conditions for both the myotrophin cDNA (42°C, 5 ϫ SSPE, 10 ϫ Denhardt's, 50% formamide, 2% SDS, and 100 g/ml salmon sperm DNA) and RNA (50°C, 1.5 ϫ SSPE, 1% SDS, 0.5% BLOTTO, 50% formamide, tRNA (0.2 mg/ml), and salmon sperm DNA (0.5 mg/ml)) probes. Approximately 5 g of pure poly(A) RNA (lane a in Fig. 1B) and 2 g of "low salt wash RNA" (lane b in Fig.  1B) from 9-day-old WKY rat hearts was fractionated on the agarose gel. High stringency hybridization and wash conditions described above were used to analyze this Northern blot; experiments were repeated several times with different batches of RNA, and the results were reproducible. The multiple tissue Northern blot containing pure poly(A) RNA from various rat tissues was obtained from Clonetech (no. 7764-1) Approximately 2 g of pure poly(A) from each tissue was fractionated on the agarose gel according to Clonetech. The high stringency hybridization and wash conditions recommended by Clonetech were used to analyze this multiple tissue Northern blot and is described above. Poly(A) RNA isolated from various rat tissues in our laboratory also revealed the same results (data not shown).
Expression of Myotrophin in E. coli-Myotrophin was expressed in E. coli using the T7 promoter-based vector, pET3a (Novagen Inc.) (4). 1 The myotrophin recombinant pET3a-51 vector was introduced into BL21(DE3) LysS strain, which harbors a T7 RNA polymerase coding gene. The recombinant myotrophin was expressed by growing the E. coli cells to early log phase and was later induced with 0.1 mM isopropyl-1-thio-␤-D-galactopyranoside for 16 h. Overnight induced cells were harvested and lysed in 50 mM Tris-HCl, pH 8.0, 75 mM NaCl by freeze thawing three times. The lysed E. coli cell debris was removed by centrifugation at 10,000 ϫ g, and the soluble supernatant was used to purify the recombinant myotrophin. The soluble form of recombinant myotrophin was highly abundant in the supernatant and was separated from the rest of the E. coli proteins using a Centriprep-30 (30-kDa cutoff) Amicon cartridge. Later, the purified recombinant myotrophin was concentrated using a Centriprep-10 (10-kDa cutoff) cartridge. On a 12% Tris-Tricine SDS-PAGE, 2 the purified recombinant myotrophin * This work was supported in part by American Heart Association (Northeastern Ohio affiliate) Grant-in-aid 4847 (to N. S.) and National Institutes of Health Grant HL 47794 (to S. S.). 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 GenBank TM /EMBL Data Bank with accession number(s) U21661.
‡ To whom correspondence should be addressed. migrated as a single band at the 12-kDa region. Protein concentration was estimated using Bio-Rad protein assay reagent, and appropriate quantities of recombinant myotrophin were used in gel shift assays. The recombinant myotrophin was further tested for its immunoreactivity using native myotrophin-specific antibodies (5). Native myotrophinspecific antibodies were generated against a synthetic peptide containing the 17 amino acid residues of the T26 tryptic peptide of native myotrophin (5). Since Western immunoblot analysis clearly showed that the recombinant myotrophin was immunoreactive to myotrophinspecific antibodies (data not shown), it was used for functional studies.
Electrophoretic Mobility Shift Assays-Phorbol ester-treated human Jurkat T cell nuclear extract, B, consensus double-stranded oligonucleotide substrate (5Ј-AGTTGAGGGGACTTTCCCAGGC-3Ј), Oct-1 consensus double-stranded oligonucleotide substrate (5Ј-TGTCGAATG-CAAATCACTAGAA-3Ј), and p50 and p65 supershift antibodies were purchased from Santa Cruz Biotechnology Inc. Poly(dI-dC)⅐poly(dI⅐dC) was purchased from Pharmacia Biotech Inc. Partially purified recombinant myotrophin (4) 1 and native peptide myotrophin-specific antibody (1-3) were used in the gel shift assays. DNA-protein binding reactions were carried out in 12 mM HEPES-NaOH (pH 7.9), 4 mM Tris⅐Cl (pH 7.9), 60 mM KCl, 1 mM EDTA, and 1 mM dithiothreitol, 2 g of poly(dI-dC)⅐poly(dI⅐dC) and 10% glycerol in a final volume of 15 l. The reactions contained 10 g of Jurkat cell nuclear extract, varying amounts (1-3 l, containing 200 ng/l) of bacterially expressed recombinant myotrophin, and 10,000 cpm of end-labeled NF-B or Oct-1 binding site probe. After incubating at room temperature for 30 min, the reactions were run on a 4% PAGE using 0.25 ϫ TBE as the gel buffer and 1 ϫ TBE as the running buffer. The gel was electrophoresed at 160 volts for 2 h. Later, the gel was dried and autoradiographed overnight at Ϫ70°C. Purified myotrophin-specific antibodies (IgG) (Ref. 5) and preimmune antibodies (IgG) were preincubated with myotrophin for 1 hour in ice before the binding reactions were carried out.

Distribution of Myotrophin mRNA in Rat
Tissues-A multiple tissue Northern blot containing poly(A) RNA from various tissues of rat was obtained from Clonetech. Myotrophin-specific double-stranded cDNA probe was used to identify myotrophinspecific transcripts. The blot was hybridized and washed at very high stringency conditions. The results of the experiment are shown in Fig. 1A. In total, at least five myotrophin-specific transcripts were detected in these tissues. Among them, two high molecular weight transcripts (4.3 and 3.5 kb) were detected in almost all tissues. These transcripts were most abundant in brain and least in skeletal muscle compared to other tissues. In addition, three transcripts of 2.4, 1.8, and 1.0 kb in size were also detected in some tissues, although at different levels. These were detected more abundantly in certain tissues like testis and liver compared to other tissues. Based on its ubiquitous distribution, it appears that the myotrophin protein may be playing a very important role in the basic functions of various tissues. We have recently obtained several myotrophin cDNA clones through direct screening of a rat heart 5Ј-stretch cDNA library (Clonetech), and the preliminary characteriza-tion reveals that the size of the clone inserts correspond to the sizes of these multiple transcripts. Based on the initial nucleotide sequence data from few cDNA clones as well as data from rapid amplification of cDNA ends-polymerase chain reactions (4), 1 it appears that the heterogeneity in the length of 3Јuntranslated regions contributes to the observed heterogeneity in the multiple transcripts. The observation of multiple types of cDNA clones with different 3Ј-untranslated regions (4, 8) 1 supports the present observation of multiple transcripts in the northern hybridization experiment. Southern analysis of rat genomic DNA also suggests that myotrophin is coded by a single copy gene as revealed by our observation of a single 4.3-kilobase pair HindIII genomic DNA fragment hybridizing to the myotrophin coding region probe, 3 and hence these multiple transcripts arise from the single copy myotrophin gene. Similar types of multiple transcripts have been observed for other genes like opsin in mouse, rat, human, and frog (20).
Myotrophin Gene Expression in Rat Heart-Expression of myotrophin gene specifically in rat hearts was analyzed in more detail using Northern blot analysis. Using the coding region of myotrophin gene, both double-stranded DNA probe (lane a in Fig. 1B) as well as single-stranded antisense RNA probe (lane b in Fig. 1B) was used (see "Experimental Procedures") in different Northern blot experiments. Very high stringency hybridization and wash conditions were followed for this experiment. Initially, only the 4.3-and 3.5-kb transcripts were detected when pure poly(A) RNA was used (lane a in Fig. 1B). Since low molecular weight myotrophin transcripts were not detected significantly in this poly(A) RNA, we included a lowsalt wash step in our oligo(dT) purification procedure (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 M NaCl) before eluting the poly(A) RNA with no-salt buffer. The RNA from this low salt wash fraction was ethanol precipitated and analyzed for myotrophin-specific transcripts. Interestingly, the majority of the myotrophin-specific low molecular weight transcripts (2.4, 1.0, 0.7, and 0.4 kb) were observed mostly in the low salt-eluted RNA when compared to pure poly(A) RNA (lane b in Fig. 1B). It is possible that poly(A) tracts in these transcripts may be either shorter in length or totally devoid of and thus eluted in the low-salt buffer. The observation of several myotrophinspecific transcripts in the low salt wash RNA fraction also suggests that either these are degraded products of the myotrophin mRNAs after translation or translationally silenced mRNAs ready to be translated upon receiving the physiological signal (21,22).
Primary Structure Analysis of Myotrophin Protein-The primary structure of the myotrophin protein was analyzed thor-3 G. Adhikary, unpublished observations. oughly to locate any structural domains that would have any specific functions. For this purpose, two analyses were conducted. Initially, using the MacPattern (version 3.2) software and Prosite data base, the myotrophin amino acid sequence was analyzed to locate any possible functional domains. This analysis revealed two putative consensus phosphorylation sites for protein kinase C (TVK) and casein kinase II (TALE) (Fig.  2A). These putative domains were not reported earlier for V1 protein from rat brain (7,8). In the second analysis, the individual ankyrin repeats as well as segments of myotrophin amino acid sequence were compared against the entire Gen-Bank/EMBL protein data base using BLASTP program (6). This analysis revealed new information about the structural features of myotrophin protein. First, an additional half ankyrin repeat spanning from residues 92-107 was identified, which was not reported previously (7,8). During this analysis, a significant homology was also observed between the ankyrin repeat 2 (residues 27-58) of myotrophin and the ankyrin repeats of IB␣ proteins (9 -12) (Fig. 2B).
The ankyrin repeats of myotrophin span from amino acid residues 9 to 107 ( Fig. 2A). Myotrophin possesses two fulllength ankyrin repeats (repeat 2, 27-58 and repeat 3, 59 -91) and two incomplete (half) repeats (repeat 1, 9 -26 and repeat 4, 92-107). One incomplete repeat (9 -26) is a carboxyl half of a typical ankyrin repeat, and the other (92-107) is an amino half ( Fig. 2A). Ankyrin repeats are generally 33 amino acids in length and possess two regions: one region is highly conserved and the other is highly variable. A typical ankyrin repeat sequence is shown below, where X indicates a highly variable region compared with the rest of the conserved region: XGXT-PLHXAXXLLXXGADXXXDX.
Since the ankyrin repeats are found in various classes of proteins (cytoplasmic, nuclear, and cell surface), Hatada et al. (13) have attempted to classify these ankyrin repeats. Based on the sequence homology in the variable region of these ankyrin repeats, Hatada et al. (13) have proposed a unique subgroup of ankyrin repeats for rel and related IB transcription factors (9 -12). The BLASTP analysis of myotrophin revealed a significant homology between the ankyrin repeat 2 (residues 27-58) of myotrophin and two ankyrin repeats of IB␣ proteins (Fig.  2B). The myotrophin ankyrin repeat 2, in addition to the homology in the core consensus sequence, possesses homologous residues in the variable region similar to the IB␣ ankyrin repeat (Fig. 2B). Since the ankyrin repeats are considered as modular protein-interacting domains with sub-regions of these repeats conferring unique specificity, the observed homology between myotrophin ankyrin repeat 2 and the two ankyrin repeats of IB␣ proteins (9 -13) is considered very significant (Fig. 2B).
It is well known that the IB proteins interact with NF-B/ rel factors through its ankyrin repeats (15)(16)(17)(18)(19). Specifically, it has been shown that IB␣ ankyrin repeats bind to rel domains of NF-B subunits (p50 and p65). In addition to the ankyrin repeats, putative consensus phosphorylation sites for protein kinase C and casein kinase II, which were observed in IB␣ proteins (9 -12), were also observed in myotrophin ( Fig. 2A). However, myotrophin is only a 12-kilodalton protein with fewer ankyrin repeats than other known IB␣ proteins. The observation of IB␣ homologous ankyrin repeats and putative consensus phosphorylation sites for protein kinase C and casein kinase II in myotrophin suggested that myotrophin may be a unique IB-related protein.
Electrophoretic Mobility Shift Assays with Recombinant Myotrophin-Because of the above structural observations, electrophoretic mobility shift assays (EMSAs) were performed with Jurkat T cell nuclear extract proteins to identify whether the NF-B/rel is a target for myotrophin binding. The results are shown in Fig. 3A. Interestingly, we observed that with increasing concentrations of myotrophin, ternary complexes are formed between myotrophin and NF-B/rel. Two types of ternary complexes (lanes 5-7 in Fig. 3A) were demonstrated by PAGE. The slower migrating (SC) complexes appear to be heterotrimeric (myo-NF-B/rel), and the faster migrating (FC) complexes appear to be devoid of one of the subunits of the NF-B/rel complex (Fig. 3A). Furthermore, the preimmune serum IgG (lane 9 in Fig. 3A) did not prevent this binding, whereas the myotrophin-specific IgG (lane 8 in Fig. 3A) inhibited specifically the formation of these ternary complexes. However, myotrophin by itself did not bind to B DNA substrate probe (lane 4 in Fig. 3A). Unlike other known IB proteins, myotrophin did not inhibit the DNA binding activity of the NF-B complex; instead, it formed ternary complexes. These results were also confirmed by preliminary EMSAs with cardiac myocyte nuclear extracts (data not shown). To confirm the specificity of myotrophin interaction with the NF-B/rel complex, Oct-1 EMSAs (14) were also carried out using the same Jurkat T cell nuclear extract (Fig. 3B). With the same increasing concentrations of myotrophin, myotrophin (lanes 2-4 in Fig. 3B) did not affect any of the Oct-1⅐DNA complexes at all. These results clearly show that the specific target for myotrophin is the subunits of the NF-B/rel complex.
To further confirm the myotrophin interaction with NF-B/ rel factors, EMSAs were performed in presence of p50 and p65 antibodies. The results are shown in Fig. 4. When incubated with either p50 (lane 6) or p65 (lane 4) antibodies, the myotrophin-shifted B complexes were supershifted to slower migrating ternary complexes (SSC-50 and SSC-65 in Fig. 4), and the intensity of these complexes increased when more myotrophin was present in the reaction. These results clearly show that myotrophin-shifted protein complexes actually contain NF-B/ rel factors. It should also be noted that phorbol 12-myristate 13-acetate-induced Jurkat T cells probably contain a sufficient amount of endogenous myotrophin since myotrophin-shifted B complexes were also detected at a lower level in the control experiments (lanes 1 and 2 in Fig. 4).

FIG. 2.
A, ankyrin repeats and putative phosphorylation sites for protein kinase C and casein kinase II are highlighted on the indirectly predicted amino acid sequence of myotrophin. B, homology of myotrophin ankyrin repeat 2 to IB␣ ankyrin repeats. It should be noted that in addition to rel-associated pp40, the BLASTP analysis identified other IB␣ members (MAD3, RL/IF-1, and ECI-6) on the same ankyrin repeats 2 and 4 with similar Poisson values. DISCUSSION In the present study, we have shown that the myotrophin gene is expressed in various rat tissues and as much as seven myotrophin-specific transcripts have been detected in rat heart and in other tissues. These transcripts were most abundant in brain and least in skeletal muscle compared to other tissues. Based on its ubiquitous distribution, it appears that the myotrophin protein may be playing a very important role in the basic functions of various tissues.
Our analysis on the primary structure of the myotrophin protein also revealed the homology between one of the ankyrin repeats of myotrophin and to those of IB␣/rel ankyrin repeats. Furthermore, our analysis showed putative consensus phosphorylation sites for protein kinase C and casein kinase II in myotrophin protein, which were also observed in IB␣ proteins. The significance of these homologies were experimentally confirmed with B gel shift assays. The results of these gel shift assays clearly show that the recombinant myotrophin has the ability to interact with NF-B/rel proteins in vitro. In vivo experiments are currently being conducted to further confirm these results. Thus, these results clearly indicate that the 12-kDa myotrophin protein is a unique rel/NF-B interacting protein.
It has been very well documented that upon exposure to a variety of external stimuli, NF-B/rel proteins are involved in the rapid induction of genes whose products play a central role in the immune responses, inflammation, and cell proliferation (15)(16)(17)(18)(19). The most obvious characteristic of NF-B is its rapid translocation from cytoplasm to nucleus in response to extracellular signals. They are kept dormant in the cytoplasm by the members of the IB family of proteins. Many signals inactivate the inhibitor IB, thereby allowing the NF-B to enter nuclei and rapidly induce coordinate sets of defense-related genes. It is possible that upon exposure to chronic hemodynamic overload signals, cardiac myocytes respond through their NF-B rapid response system to alter myocardial gene expression. In the present preliminary study, we have shown by its ability to interact with NF-B in vitro that myotrophin is probably a component of such a rapid response system, which might in- rel-specific p50 and p65 antibodies were added to the appropriate B binding reactions. Bacterially expressed recombinant myotrophin was added ("ϩ" ϭ 1 l ϭ 200 ng) to the binding reactions along with appropriate antibodies, and its effect was analyzed on 4% PAGE. JNE-P, phorbol ester-treated Jurkat T cell nuclear extract; B, radiolabeled B DNA probe; myo, recombinant myotrophin; ␣-p65, antibody to p65 of NF-B complex; ␣-p50, antibody to p50 of NF-B complex; SC, myotrophin-shifted slower migrating complexes; NF-B, rel-B heterodimeric protein-DNA complexes; SSC-50, supershifted complex by ␣-p50; SSC-65, supershifted complex by ␣-p65. fluence the transcription of hypertrophy-specific genes. Based on the present study, we speculate that myotrophin is probably involved in regulating the expression of hypertrophy-specific genes in the myocardium through rel factors and B DNA sites. It should be noted that no transcription regulatory factor has been reported so far to be involved in cardiac hypertrophy. Further studies are in progress to determine the exact mechanism of action of myotrophin.