(Received for publication, July 5, 1995; and in revised form, September 5, 1995)
From the
Myotonic muscular dystrophy is an autosomal dominant defect that
produces muscle wasting, myotonia, and cardiac conduction
abnormalities. The myotonic dystrophy locus codes for a putative
serine-threonine protein kinase of unknown function. We report that
overexpression of human myotonic dystrophy protein kinase induces the
expression of skeletal muscle-specific genes in undifferentiated
BCH1 muscle cells. BC
H1 clones expressing
myotonic dystrophy kinase appear equivalent to differentiated cells
with respect to expression of myogenin, retinoblastoma tumor supressor
gene, M creatine kinase,
-tropomyosin, and vimentin. In addition,
differential display analysis demonstrates that the pattern of gene
expression exhibited by myotonic dystrophy kinase-expressing cells is
essentially identical to that of differentiated BC
H1 muscle
cells. These observations suggest that myotonic dystrophy kinase may
function in the myogenic pathway.
Myotonic muscular dystrophy (DM) ()is an autosomal
dominant myopathy of variable onset, producing pleiotropic effects
including skeletal muscle weakness and wasting, cardiac conduction
abnormalities, frontal baldness, and cataracts(1, 2) .
Severity of the disease correlates with expansion of an unstable CTG
trinucleotide repeat within the 3`-untranslated region (UTR) of the
gene for DM kinase (DMK) (3, 4, 5, 6, 7) . The
triplet repeats expand in subsequent generations, producing the genetic
phenomenon of anticipation; progeny of affected individuals generally
display earlier onset and greater severity of
symptoms(8, 9) .
The product of the DM locus has
been identified as a putative serine-threonine protein kinase that
shares homology with protein kinases A and C. The full-length cDNA
codes for a protein product with an apparent molecular mass of 71
kDa(10, 11) . ()The 71-kDa gene product
appears to be post-translationally modified to produce a second DMK
form with an apparent molecular mass of 84 kDa.
Structural
predictions from the primary sequence suggest that DMK consists of an
amino-terminal kinase domain, an
-helical coiled-coil domain, and
a carboxyl-terminal transmembrane domain(12) . The catalytic
activity of DM kinase has been verified(13) , but in vivo substrates have yet to be identified. In skeletal muscle, DMK
appears to be localized to the neuromuscular junction, while in cardiac
muscle the kinase is present in the intercalated
disc(10, 11) . DMK mRNA is present in a variety of
tissue types, including muscle, brain, and eye. Quantitation of DMK
mRNA concentrations from alleles with expanded triplet repeats in
affected individuals has proven difficult, leading to conflicting
reports as to expression of mutant
alleles(14, 15, 16) .
The disease state of
DM is likely to be related to the aberrant expression of DMK and the
subsequent failure of the kinase to act upon its proper substrates or
ectopic interaction with other substrates. In an effort to establish
whether DMK operates within a known signal transduction pathway, we
constructed stably transfected BCH1 muscle cell lines that
constitutively overexpress human DMK. DMK-expressing and untransfected
cells were cultured in the presence of 20% fetal calf serum;
differentiated BC
H1 cells were produced by mitogen
withdrawal. Total cellular RNA was isolated and subjected to mRNA
differential display analysis, a polymerase chain reaction (PCR)-based
technique useful in the identification of differentially expressed
genes(17) . The pattern of gene expression was substantially
changed in DMK-expressing clones, and the new pattern of expression was
essentially identical to that of differentiated BC
H1 cells.
Two differentially expressed gene products, consistent with a skeletal
muscle phenotype, were cloned by differential display, sequenced, and
used as Northern blot probes. Skeletal muscle-specific
-tropomyosin mRNA expression was increased, and vimentin mRNA
expression was decreased in DMK-expressing clones. Northern blot
analysis of other genes implicated in myogenesis showed that myogenin
mRNA was induced in both DMK-expressing and differentiated
BC
H1 cells. Retinoblastoma tumor supressor (Rb) mRNA was
present in untransfected cells but absent from DMK-expressing and
differentiated cells. Creatine kinase activity assays demonstrated that
DMK-expressing clones up-regulate expression of the M creatine kinase
isoform. Thus, despite the presence of mitogens, overexpression of DMK
in BC
H1 cells induces the skeletal muscle phenotype.
The first attempt to produce a stable DMK-overexpressing cell
line was unsuccessful. Stably transfected clones were produced by
transfecting BCH1 cells with a plasmid construct containing
the full-length DMK cDNA, including the 5`- and 3`-UTR. DMK mRNA and
protein were undetectable in three independently generated full-length
clones (Fig. 1, panel A, lanes 3-6, and panel B, lanes 10-13). One full-length clone,
number 11, expressed very low levels of DMK mRNA but no protein
expression could be detected. Given observations regarding the
influence of UTR sequences upon gene
expression(21, 22, 23, 24, 25) ,
and considering that the genetic lesion associated with DM is located
in the 3`-UTR of DMK, we removed the UTR to produce a coding-only
construct (coDMK). The coDMK construct was derived from the full-length
cDNA by PCR using primers that annealed to the start and termination
codon sequences. DMK coding sequences were cloned into the pcDNA3
eukaryotic expression vector. Stable transfectants were produced with
the coDMK construct and three independently isolated clonal cell lines
constitutively expressed high levels of DMK mRNA and protein (Fig. 1, panel A, lane 2, and panel
B, lane 9). Additionally, six independent DMK 3`-UTR
clones expressing high concentrations of DMK 3`-UTR mRNA were produced.
Figure 1:
DMK mRNA
and protein expression in stable full-length DMK (flDMK) and
coding-only DMK (coDMK) BCH1 clones. A, Northern
analysis. Total RNA samples from cell lines were isolated,
electrophoresed on a 2% agarose-formaldehyde gel (15 µg
loaded/lane), transferred to a nylon membrane, and hybridized with a
DMK cDNA probe. Positions of 28 and 18 S ribosomal RNAs are as noted in
figure. Lane 1, untransfected BC
H1; lane
2, coDMK clone 1; lane 3, flDMK clone 7; lane 4,
flDMK clone 8; lane 5, flDMK clone 10; lane 6, flDMK
clone 11. B, Western analysis. Protein samples from cell lines
were isolated, resolved on a SDS-polyacrylamide gel (20 µg
loaded/lane), transferred to a nylon membrane, and incubated with an
anti-DMK primary antibody. Position of the 69-kDa standard is as noted
in figure. Lane 7, untransfected BC
H1 control; lane 8, DMK 3`-UTR clone; lane 9, coDMK clone 1; lane 10, flDMK clone 7; lane 11, flDMK clone 8; lane 12, flDMK clone 10; lane 13, flDMK clone
11.
Log phase BCH1 cells growing in high mitogen (20% serum)
conditions typically exhibit a fibroblast-like morphology. In low
mitogen conditions (1% serum), cells withdraw from the cell cycle,
begin expression of muscle-specific genes, and assume an elongated,
myoblast-like appearance(26) . The coDMK transfectants were
phenotypically similar to differentiated BC
H1 myoblasts,
appearing elongated with numerous cellular outgrowths. coDMK
transfectants did not exhibit cell cycle arrest and maintained the
``differentiated'' morphology in high serum conditions. In
contrast, clones expressing DMK 3`-UTR alone retained a fibroblast-like
morphology identical to untransfected BC
H1 cells.
In order to identify changes in gene expression associated with DMK or DMK 3`-UTR overexpression, we performed differential display analysis of the stable coDMK and 3`-UTR cell lines. The patterns of gene expression in the 3`-UTR clone and the untransfected control cells were nearly identical (Fig. 2, panels A-C, lanes 2,3, 5, 6, 8, and 9). In comparison, cells expressing DMK exhibited a markedly different pattern of mRNA expression (Fig. 2, panels A-C, lanes 1, 4, and 7). Expression of numerous mRNAs were affected, apparently representing both up- and down-regulation of several genes. We initially reamplified and subcloned nine differentially expressed cDNA products. The products were both sequenced and used to probe Northern blots to verify that differential display correlated with differential expression. Six of the products were unidentifiable, either showing no significant similarity to sequences in the GenBank(TM) data base, or matching the 3` end of a partially sequenced but uncharacterized cDNA. One product represented mitochondrial sequences and was not examined further.
Figure 2:
Differential display. Autoradiographs of
mRNA differential display reactions from BCH1 cells,
resolved by electrophoresis on denaturing polyacrylamide sequencing
gels. Lanes 1, 4, and 7, DMK-expressing
cells; lanes 2, 5, and 8, DMK
3`-UTR-expressing cells; lanes 3, 6, and 9,
untransfected BC
H1. Lane 10,
untransfected/undifferentiated BC
H1; lane 11,
differentiated BC
H1; lane 12, DMK-expressing
cells. Primers used: AP-1 and T12MA (A), AP-1 and T12MC (B), AP-4 and T12MC (C), and AP-1 and T12MC (D). Positions of differentially expressed products DM114.07
(vimentin) and DM114.10 (
-tropomyosin) are
indicated.
One product, designated DM114.10, hybridized to an mRNA
unique to DMK-expressing cells (Fig. 2C and
3A, lanes 1-3). DM114.10 also hybridized to an
mRNA expressed at high concentrations in differentiated
BCH1 cells (data not shown). This product was identified as
a differentiation-dependent form of
-tropomyosin unique to
skeletal muscle. Rodents have been shown to produce two different
tissue-specific
-tropomyosin messages(27) . Fibroblasts
and smooth muscle produce a 1.1-kilobase mRNA transcript; skeletal
muscle transcripts are larger (1.2 kilobases) and possess a different
3` end produced by alternative splicing and usage of a distal
polyadenylation site. Our DM114.10 product is limited to those 3`
sequences unique to the skeletal muscle mRNA.
The clone designated
DM114.07 was identified as vimentin and recognized an mRNA present in
untransfected BCH1 cells that became diminished in
DMK-expressing cells (Fig. 2B and 3B, lanes 4-6) as well as differentiated BC
H1
cells (data not shown). Studies in rodents and humans have established
that vimentin expression is high in satellite cells and regenerating
muscle, but undetectable in mature skeletal
muscle(28, 29, 30, 31) .
Because
of the differential expression of -tropomyosin and vimentin, we
began to suspect that cells expressing DMK were initiating a portion of
a skeletal muscle differentiation program. Although the cognate
messages could not be identified, the expression patterns of two of the
differential display products, DM114.02 and DM114.03, were consistent
with a skeletal muscle phenotype. DM114.02 recognized a transcript
present in cells expressing DMK that was absent from untransfected
cells (data not shown) and had high similarity to a human cDNA isolated
from skeletal muscle (GenBank accession no. Z28822). DM114.03
hybridized to a transcript in untransfected cells that was absent in
the DMK clone (data not shown) and shared similarity with a human cDNA
from cardiac muscle (GenBank(TM) accession no. Z33441).
To
further assess the differentiation state of DMK-expressing cells, we
examined expression of two genes known to be implicated in myogenic
development: myogenin (32) and retinoblastoma
(Rb)(33, 34) . A myogenin cDNA probe detected high
levels of myogenin mRNA induced in both DMK-expressing and
differentiated cells (Fig. 4, A and B). Rb
mRNA expression was affected in a reciprocal fashion; it was abundant
in untransfected cells but decreased to undetectable levels in
DMK-expressing and differentiated BCH1 cells (Fig. 4, C and D).
Figure 4:
Northern blots of myogenin and Rb mRNA.
Myogenin and Rb cDNA probes were labeled and hybridized to total RNA
samples from BCH1 cells (15 µg loaded/lane). Positions
of 28 and 18 S ribosomal RNAs are as noted in figure. A,
myogenin probe. Lane 1, untransfected BC
H1; lane 2, DMK-expressing cells; lane 3, DMK
3`-UTR-expressing cells. B, myogenin probe. Lane 4,
untransfected/undifferentiated BC
H1; lane 5,
differentiated BC
H1. C, Rb probe. Lane 6,
untransfected BC
H1; lane 7, DMK-expressing cells; lane 8, DMK 3`-UTR-expressing cells. D, Rb probe, lane 9, untransfected/undifferentiated BC
H1; lane 10, differentiated
BC
H1.
Creatine kinase (CK) isoforms serve as markers of skeletal muscle differentiation(35, 36, 37) . Undifferentiated myogenic cells predominantly express the B isoform (Fig. 5, lane 1). Following myogenesis, M creatine kinase becomes the major isoform. We perfomed CK activity assays on DMK-expressing clones and observed that with respect to CK activity, DMK-expressing cells appeared to be equivalent to differentiated cells; M creatine kinase activity was strongly increased (Fig. 5, lanes 2 and 3).
Figure 5:
Creatine kinase isoform assays. Cell
lysates (1-4 µg) were resolved by agarose electrophoresis
isoenzyme gels. Lane 1, untransfected/undifferentiated
BCH1; lane 2, differentiated BC
H1; lane 3, DMK-expressing cells.
Finally, we performed differential
display on RNA from untransfected/undifferentiated, differentiated, and
DMK-expressing cells. The patterns of mRNA expression shown by
DMK-expressing and differentiated cells were nearly identical (Fig. 2D, lanes 11 and 12), and both
were substantially different from the pattern of mRNA expression
detected in untransfected/undifferentiated cells (Fig. 2D, lane 10).This observation suggests
that many of the changes in gene expression associated with DMK
overexpression are similar to those induced during differentiation in
BCH1 cells.
We have not determined whether the apparent
myogenic effect of DMK overexpression is a specific one, or if it is
simply a result of the nonspecific activity of an ectopically expressed
protein kinase. However, overexpression of protein kinase A or C, the
two kinases that share the greatest homology with DMK, actually inhibit
muscle-specific transcription(38, 39) . Although no
protein kinase has yet been conclusively implicated in the control of
myogenesis, it is likely that kinase cascades are involved in the
establishment and maintainance of the differentiated state in
muscle(40) . It has been speculated that DMK may influence
muscle development through an effect upon myogenic gene
products(41) . Recent observations by Meixell et
al. suggest that myogenin can serve as an in vitro substrate for DMK. Ultimately, however, any formal model will rely
upon identification of the actual in vivo substrate. It is
interesting to note that DMK-expressing cells maintain a differentiated
phenotype while actively dividing in high mitogen conditions,
suggesting that cell cycle arrest is not a prerequisite for
differentiation in these clones.