From the
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.
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)
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).
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. [
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.
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.
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
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).
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.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, (
)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).
(
)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).
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.
-
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.
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.
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.
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).
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).
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).
Table: Nonmuscle myosin heavy chain insertions
/EMBL Data Bank with accession
number(s) U15618, U15693, U15716, U15765, and U15766.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.