©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Neuronal Cell Expression of Inserted Isoforms of Vertebrate Nonmuscle Myosin Heavy Chain II-B (*)

Kazuyuki Itoh (§) , Robert S. Adelstein (¶)

From the (1)Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1762

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous work has demonstrated that unique isoforms of nonmuscle myosin heavy chain II-B (MHC-B) are expressed in chicken and human neuronal cells (Takahashi, M., Kawamoto, S., and Adelstein, R. S.(1992) J. Biol. Chem. 267, 17864-17871). These isoforms, which appear to be generated by alternative splicing of pre-mRNA, differ from the MHC-B isoform present in a large number of nonmuscle cells in that they contain inserted cassettes of amino acids near the ATP binding region and/or near the actin binding region. The insert near the ATP binding region begins after amino acid 211 and consists of either 10 or 16 amino acids. The insert near the actin binding region begins after amino acid 621 and consists of 21 amino acids. Using a variety of techniques, we have studied the distribution and expression of the inserted MHC-B isoforms. In the developing chicken brain, mRNA encoding the 10-amino acid insert gradually increases after embryonic day 4, peaks in the 10-14-day embryo, and then declines. In contrast, the mRNA encoding the 21-amino acid insert appears just before birth and is abundantly expressed in the adult chicken cerebellum.

There is a marked species difference between the distribution of the inserted isoforms in adult tissues. The mRNA encoding MHC-B containing the 10-amino acid insert near the ATP binding region is expressed at low levels in the adult chicken brain, but makes up most of the MHC-B mRNA expressed in the human cerebrum and approximately 90% of MHC-B in the human retina. It is also expressed in neuronal cell lines. The mRNA encoding MHC-B containing the 21-amino acid insert is abundantly expressed in the chicken cerebellum and human cerebrum, but is absent from the retina and cell lines. Employing human retinoblastoma (Y-79) and neuroblastoma (SK-N-SH) cell lines, an increase in expression of mRNA encoding the 10-amino acid inserted isoform was seen following treatment by a number of agonists or by serum deprivation. In each case, expression of the inserted MHC-B isoform correlated with cell differentiation (neuronal phenotype) and inhibition of cell division. Using a rat pheochromocytoma cell line (PC12), we found that prior to treatment with nerve growth factor (NGF), there was no evidence for either inserted isoform, although noninserted MHC-B was present. NGF treatment resulted in the appearance of mRNA encoding MHC-B containing the 10-amino acid insert, concomitant with neurite outgrowth. Both the inserted isoform and neurites disappeared following withdrawal of NGF, showing that the appearance and disappearance of the neurites is coupled to the inclusion and exclusion of this MHC-B insert.


INTRODUCTION

Vertebrate nonmuscle myosin II is a ubiquitous cytoskeletal protein composed of two heavy chains (approximately 200 kDa) that are noncovalently bound to two pairs of light chains (approximately 15-20 kDa) (Cheney et al., 1993). This two-headed conventional form of myosin is present in all eukaryotic cells and appears to play a role in cytokinesis (Fukui et al., 1990; Schroeder, 1976), cell motility (Warrick and Spudich, 1987), secretion (Choi et al., 1994; Ludowyke et al., 1989) and receptor capping (Pasternak et al., 1989). To date, all vertebrates studied appear to contain at least two different isoforms of the nonmuscle myosin II heavy chain (MHC), ()referred to as MHC-A and MHC-B (Kawamoto and Adelstein, 1991; Simons et al., 1991). The cDNA sequence encoding both isoforms in avians (Shohet et al., 1989; Takahashi et al., 1992) and humans (Saez et al., 1990; Simons et al., 1991; Phillips et al., 1995) shows a greater similarity across the species for the same isoform than for the two different isoforms within the species. The genes encoding the human isoforms have been localized to two different chromosomes, 22q11.2 for MHC-A (Saez et al., 1990; Simons et al., 1991) and 17p13 for MHC-B (Simons et al., 1991), and expression of the two isoforms has been shown to be regulated in a tissue-dependent manner (Katsuragawa et al., 1989; Kawamoto and Adelstein, 1991; Simons et al., 1991). For example, the expression of MHC-B relative to MHC-A is higher in brain and testes, whereas MHC-A is expressed more abundantly in spleen and intestinal cells (Kawamoto and Adelstein, 1991).

The cloning of the cDNA encoding MHC-B from a chicken brain library provided evidence for two cassettes of inserted amino acids in MHC-B (Takahashi et al., 1992). Results from S-1 nuclease and the cloning of RT-PCR products showed that two different inserted nucleotide sequences were spliced into the MHC, one encoding 10 amino acids starting after Pro-211 and one encoding 21 amino acids starting after Asp-621 (see Fig. 1). We propose calling the insert that occurs near the 25-50-kDa domain boundary of nonmuscle MHC-B, insert B1 (the 30-nucleotide insert at this location would be insert B1a and the 48-nucleotide insert, B1b) and the insert near the 50-20-kDa domain boundary, insert B2. MHC-B lacking an insertion would be referred to as noninserted MHC-B (see Fig. 1).()mRNA containing one or both of these sequences was found in chicken brain and spinal cord, but not other tissues. Similar inserts were cloned using RT-PCR from human brain mRNA. In all cases, the noninserted MHC-B was also present (Takahashi et al., 1992). The recent finding that human MHC-B genomic DNA contains exons encoding these inserts is consistent with their expression being due to alternative splicing of pre-mRNA.()Of particular note was the presence in the B1 insert of the sequence -Ser-Pro-Lys-, a consensus sequence for proline-directed kinases (Davis, 1993; Kemp and Pearson, 1990). The amino acids comprising insert B1, located near the ATP binding region, map to an area that was not resolved in the crystal structure of chicken fast skeletal muscle myosin S-1 (Rayment et al., 1993). Likewise, insert B2, located near the actin binding region, was also poorly visualized. These regions are also referred to as loop 1 and loop 2, respectively (Spudich, 1994).


Figure 1: Diagrammatic representation of the 200-kDa MHC showing the location of the 10- (B1a) and 16- (B1b) amino acid insert and the 21- (B2) amino acid insert. The head region of the MHC contains three proteolytic-sensitive regions at the 25-50 kDa, 50-20 kDa, and 20 kDa-rod domains. The sequences shown below for chicken and human are from Takahashi et al. (1992) as well as this report. The Xenopus sequence is from Bhatia-Dey et al. (1993). The underlined amino acids indicate the peptides used to generate antibodies to the inserted regions.



In this work, we first report studies on the temporal expression of both insertions during development of the chicken brain. We then survey the pattern of expression in mammalian tissues and cells with respect to each insertion. Finally, we show for a number of neuronal cell lines that insert B1a and B1b (see Fig. 1) can be induced following treatment with specific agonists known to promote cell differentiation. Preliminary reports of this work have appeared (Itoh and Adelstein, 1993, 1994; Itoh et al., 1994).


EXPERIMENTAL PROCEDURES

Materials

Nerve growth factor (NGF) and all culture media were obtained from Life Technologies, Inc. Butyrate and dibutyryl cyclic AMP were purchased from Sigma. Human cell lines, with the exception of PC12 cells, were obtained from the American Type Culture Collection (Rockville, MD), and cells were grown in the appropriate media as per the instructions. Rat pheochromocytoma PC12 cells were a generous gift from Dr. Mari Oshima (NICHD, Bethesda, MD) and were grown as monolayers in 75 cm tissue culture flasks at 37 °C in 5% CO. The culture medium (Dulbecco's modified Eagle's medium) was supplemented with 7% fetal bovine serum (FBS), 7% horse serum, 100 µg/ml streptomycin, and 100 units/ml penicillin. The cells were split, usually at a 1:6 ratio, each week, and the medium was changed twice between splits (Oshima et al., 1991). Human retinoblastoma Y-79 cells (Kyritsis et al., 1984) were maintained in suspension culture in RPMI 1640 with 10% FBS. For monolayer culture, cells from suspension were gently dissociated, diluted with culture medium (Dulbecco's modified Eagle's medium with 10% FBS) and seeded onto six-well culture plates (Falcon, Lincoln Park, NJ) that had been coated previously with a 0.2 mg/ml solution of poly-D-lysine (Sigma) for 10 min at room temperature (Campbell and Chader, 1988). Cells were grown as a monolayer for 1 day, prior to addition of butyrate or dibutyryl cAMP, then cultured for an additional period of time, usually for 4 days. Human tissue samples were procured, with appropriate permission, following autopsy in the National Cancer Institute (Bethesda, MD) or National Disease Research Interchange (NDRI) (Philadelphia, PA).

Preparation of RNA and RNA Blot Hybridization Analysis

Total RNA was prepared from cultured cells and human tissues by the method of Chomczynski and Sacchi(1987) and analyzed as previously reported (Simons et al., 1991).

RT-PCR, Competitive PCR, and Subcloning of PCR Products

The oligonucleotide primers and probes were synthesized using a Biosearch Model 8700 DNA Synthesizer (Biosearch Inc., San Rafael, CA). RT-PCR was carried out using a GeneAmp RNA PCR kit (Perkin Elmer). To assess the ratio of the expressed levels of the inserted form of MHC-B to the noninserted form of MHC-B, the competitive PCR method (Siebert and Larrick, 1992) was used. The primer sets were as follows. For human and rat: insert B1 (30 nucleotides) 5` sense primer = 5`-AGGAAGAAAGGACCATAATATTCCT-3` (human) or 5`-GAATTCGAAAGGACCATAATATTCCT-3` (rat); 3` antisense primer = 5`-GAGAAACCTGTAGTTATTAAATCCT-3`; insert B2 (63 nucleotides) 5` sense primer = 5`-TCAGAAACCTCGACAATTAAAA-3`; 3` antisense primer = 5`-CTTGGTTTTATATGCGGAGCCAAAA-3`. For chicken: insert B1 (30 nucleotides) 5` sense primer = 5`-AGGAAGAAAGGACCATAATATTCCT-3`; 3` antisense primer = 5`-TAAAAATCTGTAATTGTTAAATCCT-3`; insert B2 (63 nucleotides) 5` sense primer = 5`-AAGACCTGCAAATCCTCCTGGTGTG; 3` antisense primer = 5`-CTT-GGTCTTGTATGCAGAGCCAAAA-3`. Note that these primers flank each of the insert regions, which permits quantitation of the inserted and noninserted products.

One µg of total RNA from various cells or tissues was reverse-transcribed using random hexamers and cloned Moloney murine leukemia virus reverse transcriptase, and the resulting cDNA was amplified by PCR using a thermal cycler (Perkin Elmer). The reaction profile included 35 cycles of denaturation at 95 °C for 1 min and annealing and extension at 65 °C (human and chicken samples) or 60 °C (rat samples) for 2 min. [-P]dCTP (DuPont NEN) was used for labeling the PCR products for quantitation. The radioactive bands were excised from the agarose gel, counted using the Cerenkov method and quantitated as described previously (Siebert and Larrick, 1992). RNA samples which were subjected directly to PCR amplification yielded no significant product, indicating negligible contamination with genomic DNA. The PCR products were separated by agarose gel electrophoresis and were cloned into the EcoRI sites of pBluescriptII SK(-) or Srf1 sites of pCRscript SK(+) (Stratagene, La Jolla, CA) and sequenced with Sequenase enzyme kits (U. S. Biochemical Corp., Cleveland, OH) using the dideoxy chain termination method (Sanger et al., 1977).

Southern Blotting

PCR products in the agarose gel were transferred to a Nytran membrane (Schleicher & Schuell) and probed with an appropriate oligonucleotide labeled with bacteriophage T4 kinase (Life Technologies, Inc.) and [-P]ATP (ICN Radiochemicals, Costa Mesa, CA). Hybridization was carried out in the presence of 10% dextran sulfate at 42 °C for 16 h and washed according to the manufacturers' directions with a final wash in 0.1 SSC at 42 °C. The oligonucleotide probe sequences were as follows: 30-nucleotide insert probe: 5`-ATCGCCTAAACCAGTGAAACACCAG-3`; 48 nucleotide insert probe: 5`-AGTGGATCCCTGTTGTAT-3`; 63-nucleotide insert probe: 5`-TGTTTCTGGTCTTCATGAGCCACCA-3`.

Antibody Production

Peptides of 10 or 12 amino acids were synthesized based on the deduced amino acid sequences of the 10-amino acid insert, the 21-amino acid insert (see Fig. 1) and the carboxyl-terminal portion of human nonmuscle MHC-B (SDVNETQPPQSE) (Phillips et al., 1995) and human nonmuscle MHC-A (GKADGAEAKPAE) (Saez et al., 1990). The peptides were conjugated to keyhole limpet hemocyanin (Calbiochem) with glutaraldehyde (Sigma), and rabbits were immunized and bled by the Berkeley Antibody Company (Richmond, CA). Rabbit antiserum was purified using the appropriate peptide antigen affinity column by previously reported methods (Kelley et al., 1992).

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting

Extracts of various cells and tissues were prepared as described previously (Kelley et al., 1992). Briefly, cells or tissues were washed twice with Ca,Mg-free phosphate-buffered saline, homogenized manually in extraction buffer (500 mM NaCl, 25 mM Tris-HCl (pH 7.5), 50 mM sodium phosphate, 5 mM EDTA, 5 mM EGTA, 10 mM ATP, 5 mM dithiothreitol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin) using a glass homogenizer immersed in ice. The samples were sedimented to remove insoluble material, normalized according to their protein content as determined by the bicinchoninic acid method (Wiechelman et al., 1988), and subjected to electrophoresis in SDS-5% polyacrylamide with 0.065% bisacrylamide gels (Laemmli, 1970). To further separate the MHC-B2 isoforms in cell and tissue extracts, electrophoresis was carried out on SDS-5% polyacrylamide gels until the bromphenol blue dye front reached the bottom of the gel. At this time, another aliquot of dye was applied to the wells, and electrophoresis was continued until the second dye front reached the bottom of the gel. Under these conditions, MHC isoforms are resolved approximately one-third of the way from the bottom of the gel (Kawamoto and Adelstein, 1991). The separated proteins were transferred, using the XCELL transfer system (Novex, San Diego, CA) or MilliBlot transfer system (Millipore, Bedford, MA), to a supported nitrocellulose membrane (Schleicher & Schuell). Immunoblotting was carried out using the ProtoBlot II AP system (Promega, Madison, WI) or a Vectastain avidin-biotin phosphatase system (Vector, Burlingame, CA).

Miscellaneous Methods

Purified bovine brain myosin was a gift of Dr. Robabeh Moussavi (NHLBI). Cells were photographed using phase contrast microscopy with an attached camera (Olympus) employing a green filter.


RESULTS

Expression of Inserted mRNA during Chicken Brain Development

The two insertions were originally discovered during the cloning of MHC-B using a chicken brain cDNA library (Takahashi et al., 1992). We, therefore, studied the expression of each insert during chicken brain development. We radiolabeled RT-PCR products using [-P]dCTP and quantitated the ratio between the inserted and noninserted MHC-B transcripts as shown in Fig. 2. MHC-B1a yielded a product of 350 bp which constituted 14% of the total MHC-B mRNA at the earliest time point studied, embryonic day 4 (Fig. 2, top, stippled columns, and lower left, lane 1). Noninserted MHC-B yielded a product of 320 bp. The inserted isoform increased after embryonic day 4 and peaked in the 10 day embryo (Fig. 2, top and lower left panels, lane 5), at which time the rate of cerebellar development is maximal (Pearson, 1972). The mRNA encoding this isoform was more abundantly expressed in the cerebellum than in the cerebrum during embryonic development (data not shown). It is also more abundant in the cerebellum of chicken neonatal and adult brain (see Fig. 2. top; compare the stippled columns of lanes 8 and 9 with those of lanes 10 and 11, and bottom left, lanes 8-11). Below, we show that the adult mammalian cerebrum contains a larger percentage of insert B1a.


Figure 2: RT-PCR analysis of inserted sequences in the embryonic, neonatal, and adult chicken mRNA. The stippled columns indicate the B1a insert (30 nucleotides) and the striped columns the B2 insert (63 nucleotides (nt)). % of insert compares the amount of the B1a insert (lower left panel) at 350 bp to the total MHC-B (320 + 350 bp). The same is true for the B2 insert (lower right panel), where the inserted product is 433 bp and noninserted is 370 bp. The lower panels are 2% agarose gels stained with ethidium bromide. E, day of embryonic development; N1, day after hatching; Ad, adult; cl, cerebellum; cr, cerebrum; M, markers; C, no mRNA included for RT-PCR. The radioactive products were quantitated as described under ``Experimental Procedures'' and used for construction of the bar diagram. The bars show the average of three different experiments using different preparations of mRNA.



Fig. 2(top and lower right panels) shows that mRNA splicing to produce insert B2 (63-nucletotide insert) first appears in the 18-day embryo (lane 7) and that it increases after hatching. This insert is abundantly expressed in the adult chicken cerebellum (Fig. 2, top, lane 9, striped column, and lane 9, lower right panel). Thus, the two alternatively spliced isoforms differ in their time of appearance during chicken brain development.

Expression of the Inserted Isoforms in Mammalian Tissues and Cell Lines

We next quantitated the two inserted isoforms relative to the noninserted isoform in human tissues and cell lines using competitive RT-PCR. The final products were separated by agarose gel electrophoresis (Fig. 3) and blotted onto nylon membranes, and their identity was confirmed using the appropriate oligonucleotide probe (shown in panels below the agarose gel). Fig. 3(left) shows that insert B1a is the major MHC-B isoform in human cerebrum (lane 1) and retina (lane 4) and is approximately 30% of the total MHC-B in the Y-79 retinoblastoma cell line (lane 6). The noninserted MHC-B isoform is predominant in human cerebellum, spinal cord, and the neuroblastoma cell line SK-N-SH (lanes 2, 3, and 5). Note that the glioblastoma cell line contains no inserted mRNA (lane 7), consistent with the idea that expression of the inserted isoform is found in neuronal cells (see also ). The right panel of Fig. 3shows that insert B2 is present in the adult human cerebrum (313 bp) and, to a much lesser extent, in the human cerebellum. It is absent from the other cells surveyed.


Figure 3: RT-PCR analysis of inserted mRNA from human adult tissues and cell lines. The upper panels show ethidium bromide stain following electrophoresis in a 2% agarose gel. Below each panel is an autoradiogram of a Southern blot of the gel analyzed using a radioactive probe hybridizing to insert B1a (30-nucleotide (nt) insert, 350-bp product) (left) or insert B2 (63-nt insert, 313-bp product) (right). Noninserted MHC-B mRNA yields a product of 320 bp (left) and 250 bp (right). Sources of mRNA: lanes 1, human cerebrum; 2, human cerebellum; 3, human spinal cord; 4, human retina; 5, SK-N-SH (neuroblastoma cell line); 6, Y-79 (retinoblastoma); 7, U-138MG (glioblastoma); 8, no added mRNA.



summarizes the inserted sequences detected using competitive RT-PCR. The table shows, surveying a number of species, that only neuronal tissue contains inserted sequences. Previous studies showed no evidence for insert B1 and B2 in a variety of tissues including adult adrenal, kidney, and spleen (Takahashi et al., 1992). In contrast to the results using tissues, in which expression appears to be limited to neuronal cells, we have recently found that the Caco-2 cell line (human colon adenocarcinoma) expresses the B1a insert. On the other hand, the distribution of insert B2 is more restricted than B1. Although insert B1 is present in a number of different neuronal cell lines (see ), insert B2 has not been found in any cell line surveyed to date.

Detection of Inserted Isoforms Using Peptide-specific Antibodies

Having quantitated the relative amounts of MHC-B mRNA encoding the two different inserted isoforms, it was of interest to see if the protein products could also be detected. We made use of antibodies raised to the two peptides underlined in Fig. 1, as well as to peptides synthesized to duplicate the carboxyl-terminal sequence of MHC-A and MHC-B (see ``Experimental Procedures''). These antibodies react with both human and bovine brain MHCs, which is consistent with our findings that the amino acid sequences are identical in the relevant regions (data not shown). Fig. 4A is an immunoblot of extracts from a human glioblastoma cell line and bovine cerebrum as well as of purified bovine brain myosin (Panel 1, lanes 1, 2, and 3, respectively) that was first probed with antibodies raised to the human B1a-inserted sequence. The antibodies detect a band at 200 kDa in lanes 2 and 3, but not in lane 1. This is consistent with mRNA encoding this insert being absent from glioblastoma cells and its presence in the latter two samples. To confirm the negative control using glioblastoma cells, an equivalent sample was treated with antibodies raised to the carboxyl-terminal sequence of human MHC-B, and the second panel (lane 1) confirms the presence of noninserted MHC-B in these cells. Treatment of the same blot shown in lanes 2 and 3 (left) with the antibodies raised to the carboxyl-terminal sequence increased the intensity of the band already present due to the inserted antibody (see Fig. 4A, right panel) and is consistent with MHC-B1a and noninserted MHC-B comigrating in these gels.


Figure 4: Immunoblot analysis of myosin heavy chain isoforms using antibodies generated to inserted and carboxyl-terminal peptide sequences. A, immunoblot to detect MHC-B1a insert. Antibodies raised to the B1a peptide (see underline in Fig. 1) were used to probe (left panel) lane 1, an extract from a human glioblastoma cell line (U138MG); lane 2, an extract from bovine cerebrum; lane 3, purified bovine brain myosin. The right panel was probed with antibodies raised to the carboxyl-terminal sequence of MHC-B. Lane 1, glioblastoma extract, lanes 2 and 3 are the same blot probed in the left panel, now reprobed with the antibodies to the carboxyl-terminal sequence. B1a indicates the MHC containing the 10-amino acid insert and B the noninserted MHC. B (three left panels), lane 1 is an immunoblot of an extract from a neuroblastoma cell line (SK-N-MC) and lane 2 is from adult human cerebellum. Three sets of immunoblots are shown. The first set was probed with antibodies raised to the B2 peptide (see underline in Fig. 1). The middle blot is the same blot as the one on the left, but was now also probed with antibodies raised to the carboxyl terminus MHC-B peptide. A single band (B) is seen in the neuroblastoma lane and a doublet (B and B2) is seen in the cerebellar lane. Lane 3 is a different blot that was first probed with antibodies raised to the MHC-B carboxyl-terminal sequences and then with antibodies raised to MHC-A carboxyl-terminal sequences. B2, B, and A indicate the MHC-B isoform containing the 21-amino acid insert, the MHC-B isoform without the insert, and the MHC-A isoform, respectively. B (right panel), lane 1 is an immunoblot of an extract from human cerebrum, and lane 2 is from human cerebellum. Both lanes are probed with antibodies raised to the carboxyl terminus MHC-B peptide, which detects both MHC-B2 (B2) and noninserted MHC-B (B).



Fig. 4B (left panels) is an immunoblot that was carried out following SDS-polyacrylamide gel electrophoresis of extracts from a human neuroblastoma cell line (SK-N-MC) and human cerebellum. Three panels are shown, each containing an extract from the cell line and the cerebellum. These immunoblots were treated with the following antibodies. The first panel was treated with antibodies raised to a 12-amino acid peptide synthesized based on insert B2 (see Fig. 1). The panel shows that these antibodies recognize an isoform of MHC-B present in an extract prepared from human cerebellum (lane 2, B2), but not present in the neuroblastoma cell line (lane 1). The same blot, shown as the middle panel, was then probed with antibodies raised to the carboxyl terminus of MHC-B. A single band due to the noninserted MHC-B isoform can now be seen in the neuroblastoma extract (middle panel, lane 1), and a doublet is now seen in the cerebellar extract (lane 2). The new band detected in the cerebellar extract is the faster migrating one and is also the noninserted isoform of MHC-B. The third panel was probed with antibodies raised to the carboxyl-terminal sequence of MHC-B and then with antibodies raised to the carboxyl-terminal sequence of MHC-A. A doublet can be seen in the neuroblastoma extract, and three bands can be seen in the cerebellar extract. The antibodies to MHC-A detect only the fastest migrating band (labeled A). This blot demonstrates the presence in an extract of human cerebellum of three MHC isoforms. The slowest migrating isoform is the inserted isoform MHC-B2. The antibodies to MHC-B (carboxyl terminus sequence) cross-react with both MHC-B2 as well as the noninserted isoform, as would be expected since they share the same carboxyl-terminal residues.

Fig. 4B (right panel) is an immunoblot of a human brain extract from the cerebrum and cerebellum. The SDS-polyacrylamide gel electrophoresis was carried out to separate the inserted and noninserted isoforms as in the left panel, and the blot was probed with antibodies to the carboxyl terminus peptide of MHC-B. The blot shows that, in the human cerebrum, there is more of the inserted MHC-B2 than the noninserted isoform. In the cerebellum, the amount of both isoforms is approximately equal as determined by immunoblot analysis.

Induced Expression of the 30-Nucleotide Insert in Neuronal Cell Lines

We next attempted to increase expression of the inserted mRNAs in cell lines by treating them with agonists that are known to promote neuronal cell differentiation. Fig. 5shows the results of a competitive RT-PCR to detect mRNA encoding noninserted MHC-B (320-bp product) and MHC-B1a (350-bp product). A Southern blot probed with a P-labeled oligonucleotide which hybridizes to the B1b insert (top) and B1a insert (bottom) is shown below the ethidium bromide-stained gel. Lane 1 confirms the high content of B1a mRNA in human retina. Whereas there is no difference in the amount of inserted mRNA between Y-79 cells grown in suspension or attached to polylysine-coated plates (lanes 2 and 3), stimulation of the attached cells by treatment with 2 mM butyrate results in an increase of B1a mRNA compared to noninserted MHC-B mRNA (lane 4). In addition, there is a new 368-bp product which was subcloned and sequenced and found to contain insert B1b. As shown in Fig. 1, the cDNA-derived amino acid sequence of this new insert (human inserted MHC-B1b) is similar to the derived sequence for Xenopus MHC-B from this same region (Bhatia-Dey et al., 1993, see ``Discussion''). As expected, the sequence of the 350-bp product contained insert B1a. Treating Y-79 cells with 2 mM dibutyryl cAMP for 4 days resulted in a small increase in B1a mRNA, but only a trace of B1b mRNA (Fig. 5, A and B, lane 5). When the neuroblastoma cell line SK-N-SH was grown in 1% serum, conditions that favor neuronal differentiation, there was increased expression of insert B1a, compared to when they are grown in 10% fetal bovine serum (Fig. 5, A and B, compare lanes 6 (1% serum) and 7 (10% serum)).


Figure 5: RT-PCR of cell lines induced to express the B1 insert. A, ethidium bromide stain following electrophoresis of RT-PCR products in a 2.5% agarose gel. B, autoradiogram of a Southern blot of the gel hybridized with an oligonucleotide probe specific to (top) insert B1b (48-nucleotide insert, 368-bp product) and (bottom) insert B1a (30-nucleotide insert, 350-bp product) (see ``Experimental Procedures''). Sources of mRNA: lanes 1, human retina; 2, Y-79, human retinoblastoma cell line (suspension); 3, Y-79, attached to polylysine-coated plates; 4, attached Y-79 cells, stimulated by 2 mM butyrate for 4 days; 5, attached Y-79 cells, stimulated by 2 mM dibutyryl cAMP for 4 days; 6, SK-N-SH, neuroblastoma cell line, in 1% FBS; 7, same as 6, but 10% FBS; 8, no added mRNA. M indicates the marker lane.



Of note was the observation that treatment of Y-79 cells with butyrate resulted in a differentiated phenotype. They stopped dividing and flattened out (data not shown). Similar changes were seen in the neuroblastoma cells grown in 1% FBS. These changes were reversible following withdrawal of butyrate from Y-79 cells and restoration of 10% FBS to the neuroblastoma cells. Concomitantly, the ratio of the inserted mRNA to noninserted mRNA was also restored to prestimulation levels.

The rat pheochromocytoma cell line (PC12) was particularly instructive for studying changes in morphology that correlated with changes in the alternative splicing of MHC-B mRNA. Fig. 6shows the results of treating PC12 cells with 50 ng/ml NGF. At the end of 1 week, the cells stopped dividing, and incipient neurite outgrowth could be seen (Fig. 6, 1W). By 3 weeks, there is a massive outgrowth of neurites (Fig. 6, 3W), all of which disappear in 1 week following withdrawal of NGF. mRNA analysis of these cells using RT-PCR and Southern blotting reveals that, prior to stimulation with NGF, there is only mRNA encoding noninserted MHC-B (Fig. 7, lane 0, 321 bp). Following stimulation with NGF for 1 week, there is evidence for the presence of B1a mRNA (Fig. 7B, 1W). With continued stimulation by NGF, the amount of inserted isoform increases (Fig. 7, 3W). Withdrawal of NGF results in the disappearance of the inserted mRNA (Fig. 7, 4W) as well as the rounding up of the PC12 cells (Fig. 6). mRNA blot analysis during NGF treatment and after withdrawal showed no significant change in the amount of MHC-B transcript (data not shown). Thus, the reversible appearance of insert B1a mRNA correlates with neuronal differentiation of rat PC12 cells.


Figure 6: Phase contrast micrographs of PC12 cells treated with NGF. ( 174) Top left, prior to treatment with NGF; top right, after 1 week of 50 ng/ml NGF; bottom right, after 3 weeks of 50 ng/ml NGF; bottom left, 1 week after withdrawal of NGF. Note the incipient neurite outgrowth after 1 week and the massive outgrowth after 3 weeks.




Figure 7: RT-PCR of PC12 cell mRNA to detect the B1a insert. A, ethidium bromide stained gel showing noninserted (321 bp) and inserted (351 bp) PCR products. B, Southern blot analysis of the same gel hybridized with a P-labeled oligonucleotide probe specific to the B1a insert. 0, mRNA from PC12 cells prior to treatment with NGF; 1w, 1 week after treatment with NGF; 3w, 3 weeks after treatment with NGF; 4w, 1 week after withdrawal of NGF from the same cells; M, markers; SK, neuroblastoma cell line SK-N-SH mRNA (positive control for the B1a insert).




DISCUSSION

In this report, we provide evidence for the specific expression of different isoforms of nonmuscle MHC-B in neuronal cells for a number of different species. These isoforms are generated by alternative splicing of the pre-mRNA that encodes nonmuscle MHC-B. The noninserted isoform of MHC-B is present in a great many nonmuscle and muscle cells. To date, we have not found an inserted MHC-A isoform, but recently Bement et al.(1994) showed that a colon adenocarcinoma cell line, Caco-2BBe, contains a mutated form of what is apparently MHC-A, with an insert near the 25-50-kDa domain boundary (Bement et al., 1994). Fig. 1shows the two locations of the three insertions in MHC-B and compares the amino acid sequences found in chickens, humans and Xenopus cells (Bhatia-Dey et al., 1993). In the Xenopus MHC-B isoform, there is an insertion of 16 amino acids following amino acid 211 with sequence similarities to the 10- and 16-amino acid insertions found in human inserted MHC-B. However, these sequences are not confined to neuronal cells, but are present in all Xenopus cells examined to date. Moreover, in contrast to avian and mammalian neuronal cells, there is no evidence for a noninserted MHC-B isoform in Xenopus cells, nor do Xenopus cells appear to contain an insertion near to the actin binding region (Bhatia-Dey et al., 1993). Previous work has also demonstrated that 7 amino acids (-Q-G-P-S-F-S-Y-) are inserted after amino acid 211 in the avian gizzard smooth muscle MHC, which is encoded by a different gene than the nonmuscle MHC. The insertion of these 7 amino acids appears to increase the rate of in vitro movement of actin filaments over myosin heads as well as the actin-activated myosin MgATPase activity (Kelley et al., 1993). This tissue-specific smooth muscle myosin isoform is the product of alternative splicing of smooth muscle pre-mRNA (Babij, 1993; White et al., 1993).

The two inserted isoforms of MHC-B described here differ in a number of respects (). We have been unable to identify any cultured cell line to date that contains the insert following amino acid 621, although a number of neuronal cell lines contain the insert following amino acid 211. As Fig. 2demonstrates, there is a marked difference in the timing of the appearance of insert B1a mRNA, which is already being expressed in the 4-day chick embryo, and insert B2 mRNA, which does not appear until between the 15th and 18th day of embryogenesis. It is of note that Murakami et al.(1991) and Murakami and Elzinga (1992), using SDS-PAGE, were able to detect a slower migrating isoform of brain myosin that appeared just before birth in rat brain and that was also expressed in adult brain, which is most likely due to the presence of insert B2 (see Fig. 4B). Sun and Chantler(1992) have described a neuronal-specific MHC present in rat brain, which contains a unique carboxyl-terminal sequence. Although this isoform bears marked similarity in sequence to both MHC-B and MHC-A, it does not contain either insert described here.

There is also a marked difference in the distribution of inserted MHC-B isoforms between avian and mammalian species, with respect to the brain. Previously, we demonstrated that insert B2 mRNA was abundantly expressed in chicken cerebellum and could also be detected by immunoblot analysis using insert-specific antibodies (Takahashi et al., 1992). It was expressed to a lesser extent in the avian cerebrum. Insert B1a mRNA is expressed to a much smaller extent in the adult chicken brain than B2. shows that the pattern of expression is quite different in the adult mammalian brain. The insertions in mammals are expressed to a greater extent in the cerebrum compared to the cerebellum and insert B1a is more abundantly expressed than insert B2. The retina is particularly distinctive in that approximately 90% of the expressed MHC-B contains insert B1a, but it shows no expression of insert B2. Note that in general, the overall expression of MHC-B in the neuronal cells and tissues shown in is approximately the same by Northern blot analysis (data not shown).

Inspection of Fig. 1shows that a site for proline-directed kinases is preserved in amphibian and mammalian species at Ser-214. In vitro phosphorylation of this site by p34 kinase has been demonstrated for Xenopus MHC-B (Kelley et al., 1995), bovine brain myosin, and a baculovirus-expressed MHC-B containing the avian insert.()To date, only Xenopus myosin has been shown to be phosphorylated at this site in intact cells (Kelley et al., 1995). It should be noted that in vitro phosphorylation studies reveal little about the putative enzyme that is active in intact brain cells, and we are presently conducting studies with mitogen-activated protein kinase (Davis, 1993) as well as other proline-directed kinases (Kemp and Pearson, 1990).

As Fig. 1shows, the human MHC-B 10- and 16-amino acid inserts are almost identical to that found in Xenopus cells. Recently, S. Kawamoto (NHLBI) obtained a human genomic clone from this region which contains two separate exons, one of 30 nucleotides and one of 18 nucleotides, confirming the possible expression of at least three alternatively spliced isoforms (no insert, 30 nucleotides and 48 nucleotides). To date, we have seen significant quantities of the 48-nucleotide insert only in Y-79 cells treated with butyrate (Fig. 5) and another human retinoblastoma cell line (WERI-Rb-1) (data not shown).

Dibutyryl cAMP, which strongly stimulates Y-79 cell differentiation (Kyritsis et al., 1986) and increases the synthesis of melatonin (Pierce et al., 1989; Wiechelman et al., 1988), stimulated only a small increase in insert B1a expression (Fig. 5, lane 5). In contrast, butyrate, a naturally occurring four-carbon fatty acid, has been shown to inhibit growth of Y-79 cells (Kruh, 1982), induce morphological differentiation (Nakagawa and Perentes, 1987), and alter protein translation from mRNA (Kapoor et al., 1985). However, butyrate failed to increase melatonin synthesis (Deng et al., 1991; Wiechmann et al., 1990), suggesting that it acts through a different mechanism to induce morphological and biochemical differentiation. The precise mechanism of its action is not known, but it may act via induction of histone acetylation and alterations in chromatin structure and activity (Kruh, 1982). As shown in Fig. 5, butyrate markedly increases insert B1a and B1b expression, showing that different agonists have a specific effect. Neither retina nor cerebrum, which abundantly express insert B1a, show evidence for insert B1b, suggesting that its expression may be confined to retinoblastoma cells.

The experiment using PC12 cells and NGF deserves a comment. In contrast to NGF, epidermal growth factor stimulates PC12 cell conversion to another phenotype which lacks neurites and results in cell proliferation (Oshima et al., 1991). We could not detect expression of insert B1a following treatment of PC12 cells with epidermal growth factor (data not shown). On the other hand, collagen (extracellular matrix) induces neurite outgrowth from PC12 cells. The expression of insert B1a was increased, although to a lesser extent compared with NGF stimulation, in collagen-stimulated cells (see ). These findings suggest that the increase in B1a expression is associated with a particular phenotype that manifests both neurite outgrowth and inhibition of cell division in these cells.

In summary, in Xenopus cells, there is an isoform of nonmuscle MHC-B, which always includes 16 amino acids following amino acid 211 in the head region when compared to MHC-B from avian and mammalian species. This MHC-B isoform is expressed in most, if not all, Xenopus cells (Bhatia-Dey et al., 1993). In contrast, the exon(s) encoding this insert is not usually spliced into avian and normal mammalian cells, with one exception. They are spliced into neuronal cells, and most abundantly into the cells present in the mammalian human retina and cerebrum. This insert appears to play a role in terminal differentiation of neuronal cells. Its early appearance in embryonic chicken brain also suggests a role during brain development. There is a second insert of 21 amino acids following amino acid 621 in the neuronal myosin head region of avian and mammalian cells. Unlike the 10-amino acid insert, the 21-amino acid insert is not present in any cell lines, nor is it present in the retina. Both inserts B1a and B2 are abundant in the mammalian cerebrum. We are presently trying to understand the exact function of these inserts in generating a neuronal phenotype.

  
Table: Nonmuscle myosin heavy chain insertions



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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/EMBL Data Bank with accession number(s) U15618, U15693, U15716, U15765, and U15766.

§
Recipient of a Japanese Heart Foundation Fellowship.

To whom correspondence should be addressed: National Institutes of Health, Bldg. 10, Rm. 8N202, 10 Center Dr. MSC 1762, Bethesda, MD 20892-1762. Tel.: 301-496-1865; Fax: 301-402-1542.

The abbreviations used are: MHC, myosin heavy chain; NGF, nerve growth factor; RT, reverse transcriptase; PCR, polymerase chain reaction; myosin S-1, myosin subfragment-1; FBS, fetal bovine serum; bp, base pair(s).

Note that this differs from the nomenclature used in Takahashi et al. (1992), but is more consistent, since the region where insert B1 is present has been referred to as loop 1 and the region where insert B2 is present as loop 2 (Spudich, 1994).

S. Kawamoto, unpublished results.

K. Itoh and R. S. Adelstein, unpublished observation; this laboratory.


ACKNOWLEDGEMENTS

We acknowledge the excellent technical assistance of Dr. Beth Goens (NHLBI) and Jimena Maranon (NHLBI) in purifying RNA from chicken embryos as well as the helpful advice of Drs. Mari Oshima (NICHD) and Gordon Guroff (NICHD) for the PC12 cell experiment, Drs. David Klein (NICHD) and Gerald Chader (NEI) for the Y-79 cell experiment. We also thank Drs. Mary Anne Conti, Sachiyo Kawamoto, Christine A. Kelley, and James R. Sellers for critical reading of the manuscript and Catherine S. Magruder for superb editorial assistance.


REFERENCES
  1. Babij, P.(1993) Nucleic Acids Res.21, 1467-1471 [Abstract]
  2. Bement, W. M., Hasson, T., Wirth, J. A., Cheney, R. E., and Mooseker, M. S.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 6549-6553 [Abstract]
  3. Bhatia-Dey, N., Adelstein, R. S., and Dawid, I. B.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 2856-2859 [Abstract]
  4. Campbell, M., and Chader, G. J.(1988) Ophthalmic Paediatr. Genet.9, 171-199 [Medline] [Order article via Infotrieve]
  5. Cheney, R. E., Riley, M. A., and Mooseker, M. S.(1993) Cell Motil. Cytoskeleton24, 215-223 [Medline] [Order article via Infotrieve]
  6. Choi, O. H., Adelstein, R. S., and Beaven, M. A.(1994) J. Biol. Chem.269, 536-541 [Abstract/Free Full Text]
  7. Chomczynski, P., and Sacchi, N.(1987) Anal. Biochem.162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  8. Davis, R. J.(1993) J. Biol. Chem.268, 14553-14556 [Free Full Text]
  9. Deng, M. H., Coviella, I. L. G., Lynch, H. J., and Wurtman, R. J. (1991) Brain Res.561, 274-278 [Medline] [Order article via Infotrieve]
  10. Fukui, Y., De Lozanne, A., and Spudich, J. A.(1990) J. Cell Biol.110, 367-378 [Abstract]
  11. Itoh, K., and Adelstein, R. S.(1993) Mol. Biol. Cell4, 41a
  12. Itoh, K., and Adelstein, R. S.(1994) Mol. Biol. Cell5, 403a
  13. Itoh, K., Moussavi, R. S., and Adelstein, R. S.(1994) FASEB J.8, A1303
  14. Kapoor, C. L., Kyritsis, A. P., and Chader, G. J.(1985) Neurochem. Int.7, 285-294
  15. Katsuragawa, Y., Yanagisawa, M., Inoue, A., and Masaki, T.(1989) Eur. J. Biochem.184, 611-616 [Abstract]
  16. Kawamoto, S., and Adelstein, R. S.(1991) J. Cell Biol.112, 915-924 [Abstract]
  17. Kelley, C. A., Sellers, J. R., Goldsmith, P. K., and Adelstein, R. S. (1992) J. Biol. Chem.267, 2127-2130 [Abstract/Free Full Text]
  18. Kelley, C. A., Takahashi, M., Yu, J. H., and Adelstein, R. S.(1993) J. Biol. Chem.268, 12848-12854 [Abstract/Free Full Text]
  19. Kelley, C. A., Oberman, F., Yisraeli, J. K., and Adelstein, R. S. (1995) J. Biol. Chem.270, 1395-1401 [Abstract/Free Full Text]
  20. Kemp, B. E., and Pearson, R. B.(1990) Trends Biochem. Sci.15, 342-346 [Medline] [Order article via Infotrieve]
  21. Kruh, J.(1982) Mol. Cell. Biochem.42, 65-82 [Medline] [Order article via Infotrieve]
  22. Kyritsis, A. P., Tsokos, M., Triche, T. J., and Chader, G. J.(1984) Nature307, 471-473 [Medline] [Order article via Infotrieve]
  23. Kyritsis, A. P., Tsokos, M., and Chader, G. J.(1986) Anticancer Res.6, 465-474 [Medline] [Order article via Infotrieve]
  24. Laemmli, U. K.(1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  25. Ludowyke, R. I., Peleg, I., Beaven, M. A., and Adelstein, R. S.(1989) J. Biol. Chem.264, 12492-12501 [Abstract/Free Full Text]
  26. Murakami, N., and Elzinga, M.(1992) Cell Motil. Cytoskeleton22, 281-295 [Medline] [Order article via Infotrieve]
  27. Murakami, N., Mehta, P., and Elzinga, M.(1991) FEBS Lett.278, 23-25 [CrossRef][Medline] [Order article via Infotrieve]
  28. Nakagawa, Y., and Perentes, E.(1987) Ophthalmic Res.19, 205-212 [Medline] [Order article via Infotrieve]
  29. Oshima, M., Sithanandam, G., Rapp, U. R., and Guroff, G.(1991) J. Biol. Chem.266, 23753-23760 [Abstract/Free Full Text]
  30. Pasternak, C., Spudich, J. A., and Elson, E. L.(1989) Nature341, 549-551 [CrossRef][Medline] [Order article via Infotrieve]
  31. Pearson, R.(1972) The Avian Brain, pp. 74-127, Academic Press, New York
  32. Phillips, C. L., Yamakawa, K., and Adelstein, R. S.(1995) J. Muscle Res. Cell Motil., in press
  33. Pierce, M. E., Barker, D., Harrington, J., and Takahashi, J. S. (1989) J. Neurochem.53, 307-310 [Medline] [Order article via Infotrieve]
  34. Rayment, I., Rypniewski, W. R., Schmidt-Bäse, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G., and Holden, H. M.(1993) Science261, 50-58 [Medline] [Order article via Infotrieve]
  35. Saez, C. G., Myers, J. C., Shows, T. B., and Leinwand, L. A.(1990) Proc. Natl. Acad. Sci. U. S. A.87, 1164-1168 [Abstract]
  36. Sanger, F., Nicklen, S., and Coulson, A. R.(1977) Proc. Natl. Acad. Sci. U. S. A.74, 5463-5467 [Abstract]
  37. Schroeder, T. E.(1976) in Cell Motility (Goldman, R., Pollard, T., and Rosenbaum, J., eds) pp. 265-278, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  38. Shohet, R. V., Conti, M. A., Kawamoto, S., Preston, Y. A., Brill, D. A., and Adelstein, R. S.(1989) Proc. Natl. Acad. Sci. U. S. A.86, 7726-7730 [Abstract]
  39. Siebert, P. D., and Larrick, J. W.(1992) Nature359, 557-558 [CrossRef][Medline] [Order article via Infotrieve]
  40. Simons, M., Wang, M., McBride, O. W., Kawamoto, S., Yamakawa, K., Gdula, D., Adelstein, R. S., and Weir, L.(1991) Circ. Res.69, 530-539 [Abstract]
  41. Spudich, J. A.(1994) Nature372, 515-518 [Medline] [Order article via Infotrieve]
  42. Sun, W., and Chantler, P. D.(1992) J. Mol. Biol.224, 1185-1193 [Medline] [Order article via Infotrieve]
  43. Takahashi, M., Kawamoto, S., and Adelstein, R. S.(1992) J. Biol. Chem.267, 17864-17871 [Abstract/Free Full Text]
  44. Warrick, H. M., and Spudich, J. A.(1987) Annu. Rev. Cell Biol.3, 379-421 [CrossRef]
  45. White, S., Martin, A. F., and Periasamy, M.(1993) Am. J. Physiol.264, C1252-C1258
  46. Wiechelman, K., Braun, R., and Fitzpatrick, J.(1988) Anal. Biochem.175, 231-237 [Medline] [Order article via Infotrieve]
  47. Wiechmann, A. F., Kyritsis, A. P., Fletcher, R. T., and Chader, G. J. (1990) J. Neurochem.55, 208-214 [Medline] [Order article via Infotrieve]

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