From the Departments of § Internal Medicine,
¶ Molecular Biology/Oncology, and Pathology,
University of Texas Southwestern Medical Center, Dallas, Texas
75235
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ABSTRACT |
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Mitochondrial biogenesis and mitochondrial DNA
(mtDNA) replication are regulated during development and in response to
physiological stresses, but the regulatory events that control the
abundance of mtDNA in cells of higher eukaryotes have not been defined
at a molecular level. In this study, we observed that expression of the
catalytic subunit of DNA polymerase (POL
CAT)
mRNA varies little among different tissues and is not increased by
continuous neural activation of skeletal muscle, a potent stimulus to
mitochondrial biogenesis. Increased copy number for the POL
locus in
a human cell line bearing a partial duplication of chromosome 15 increased the abundance of POL
CAT mRNA without
up-regulation of mtDNA. In contrast, expression of mitochondrial
single-stranded DNA-binding (mtSSB) mRNA is regulated coordinately
with variations in the abundance of mtDNA among tissues of mammalian
organisms and is up-regulated in association with the enhanced
mitochondrial biogenesis that characterizes early postnatal development
of the heart and the adaptive response of skeletal myofibers to motor
nerve stimulation. In addition, we noted that expression of mtSSB is
concentrated within perinuclear mitochondria that constitute active
sites of mtDNA replication. We conclude that constitutive expression of the gene encoding the catalytic subunit of mitochondrial DNA polymerase is sufficient to support physiological variations in mtDNA replication among specialized cell types, whereas expression of the mtSSB gene is
controlled by molecular mechanisms acting to regulate mtDNA replication
or stability in mammalian cells.
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INTRODUCTION |
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Cells of vertebrate organisms differ markedly in their ability to utilize oxidative phosphorylation to meet cellular energy requirements, reflecting major variations in the cellular content of mitochondria and of respiratory proteins. This variation is most striking among specialized subtypes of striated myocytes (1). Mitochondria may occupy 50% or more of cell volume in cardiac myocytes of some species while type IIb skeletal myofibers have sparse mitochondria (approximately 1% of cell volume). Other subtypes of skeletal myofibers (type I or IIa) occupy intermediate positions on this spectrum. Mitochondria-rich muscle subtypes are resistant to fatigue, whereas glycolytic myofibers with few mitochondria are adapted for short bursts of high power output and fatigue rapidly with sustained activity. Cells adapted for high rates of respiration exhibit increased expression of both nuclear and mitochondrial genes encoding enzymes of oxidative phosphorylation, and enhanced mitochondrial gene expression is accompanied by amplification of the mitochondrial genome relative to nuclear DNA in mitochondria-rich muscle subtypes (2, 3). These specialized characteristics of myocytes with respect to respiratory capacity and mtDNA are established by developmental cues but can be modified in adults by changing physiological demands or disease processes (2-4).
In an effort to gain greater understanding of the molecular mechanisms
by which developmental and physiological regulation of mitochondrial
DNA (mtDNA)1 content is
achieved, we examined expression of nuclear genes encoding two proteins
required for mtDNA replication. DNA polymerase (POL
) catalyzes
replication of mtDNA, and conserved genes encoding the catalytic
subunit of POL
have recently been cloned from several eukaryotic
species, including humans (5, 6). Disruption of the POL
gene in
yeast (7) demonstrates that the enzyme has no essential functions
outside of mitochondria but is absolutely required for mtDNA
replication. A mitochondrial single-stranded DNA binding protein
(mtSSB) also is necessary for mtDNA replication in yeast (8), and
mtSSBs have been identified in several vertebrate species (9). As
assessed in cell-free reactions, mtSSB augments both fidelity and
processivity of POL
(10, 11).
In this study, we examined expression of the POLCAT and
mtSSB genes in different tissues or specialized subtypes of striated myocytes with markedly different contents of mtDNA. In addition, we
characterized changes in expression of these genes in striated muscles
responding to continuous motor nerve stimulation, a potent stimulus to
replication of mtDNA. The results show that expression of
POL
CAT mRNA is constitutive while expression of
mtSSB is strictly regulated and correlates directly with mtDNA content.
Regulated expression of mtSSB also was observed in conjunction with the up-regulation of mtDNA that occurs within the myocardium during the
first few weeks of post-natal life. Overexpression of
POL
CAT evoked by an increase in gene dosage failed to
affect the content of mtDNA. In addition, we noted that the mtSSB
protein is distributed nonuniformly within murine myogenic cells in
culture and is preferentially localized to perinuclear mitochondria
that constitute active sites of mtDNA replication (12). These data
indicate that expression of nuclear genes encoding proteins essential
for mtDNA replication is differentially, rather than coordinately,
regulated in response to signaling pathways that control mitochondrial
biogenesis in mammalian cells.
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EXPERIMENTAL PROCEDURES |
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Cloning of cDNA and Genomic Sequences Encoding mtSSB and
POLCAT--
Using primers based on the published
sequences of the rat and human mtSSB cDNAs, partial cDNAs
encoding rabbit and mouse mtSSB proteins were cloned using reverse
transcriptase-PCR. 20 µg of total heart RNA were reversed transcribed
using Superscript II (Boehringer Mannheim) by the manufacturer
recommendations. The PCR primers for cloning rabbit mtSSB cDNA
were: 1) 5
-CTNGCNACNAAYGARATGTGG-3
, and 2)
5
-ACRTTRTTYTTRTCCATRTAYTC-3
. The PCR primers for cloning mouse mtSSB
cDNA were: 1) 5
-AGGCTTGCGCGTCAGGAA-3
, and 2)
5
-TAGACTGTACATGATTTGCAAAGG-3
. Amplification products were cloned and
sequenced. The mouse and rabbit partial cDNAs (~200 nucleotides)
exhibited 94 and 85% nucleotide sequence identity, respectively, with
the published rat mtSSB sequence (9).
Animals and Tissues--
Eighteen New Zealand White rabbits were
purchased from Myrtle's Rabbitry (Thompson Station, TN). The common
peroneal nerve was stimulated continuously at 6-10 Hz as described
previously (13). Animals were sacrificed after 1, 3, 7, 14, or 21 days of stimulation (n = 3 at each time point), and tibialis
anterior (TA) muscles from stimulated and unstimulated hindlimbs were
frozen in liquid nitrogen and stored at 70 °C. From control
animals, the TA, soleus, gastrocnemius, plantaris, and extensor
digitorum longus were removed and stored in a similar manner. Pregnant
mice (strain ICR) were purchased from Harlan Sprague Dawley, Inc.
Litter sizes ranged from 8-12 pups. Littermates were sacrificed on day 1, day 5, day 10, day 15, and day 20 post-partum, and as adults (n = 4-6 at each time point). At sacrifice, the hearts
were removed, frozen in liquid nitrogen, and stored at
70 °C. All
animal protocols were reviewed and approved by the Institutional Animal
Care and Research Advisory Committee and were conducted in accordance
with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Analysis of Muscle RNA and DNA--
RNA and DNA were isolated
from muscle homogenates or cultured cells, and Northern and Southern
blots were prepared and probed by standard procedures. DNA samples were
restricted with HindIII (rabbit DNA) or EcoRI
(mouse DNA) to permit unambiguous identification of mtDNA. mRNA
samples prepared from specific human tissues were obtained from a
commercial source (CLONTECH). Species-specific radiolabeled probes were prepared by PCR or by extension of random hexamer oligonucleotides using mtSSB cDNA, PolCAT
cDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28 S
ribosomal RNA, mtDNA, or a chromosome 15 marker, D15S10, as the
template.
Production of a Polyclonal Antibody against the Rat mtSSB
Protein--
A 409-base pair fragment of the rat mtSSB cDNA,
corresponding to the full-length mature protein, was obtained through
PCR using a rat mtSSB full-length cDNA as the template.
NcoI and EcoRI restriction sites were engineered
into the 5 and 3
ends of the cDNA, respectively, and the fragment
was subcloned into the expression vector, pGEX-CS (14). Induction of
transformants with 0.1 mM isopropyl-1-thio-
-D-galactopyranoside-generated
recombinant rat mtSSB fused to glutathione S-transferase
(GST). The purification procedure included affinity chromotography
using glutathione-agarose beads (Sigma), followed by incubation with
tobacco etch virus protease (Life Technologies, Inc.) to release mtSSB
from the resin-bound GST moiety. The supernatant was concentrated with
a Centriprep unit (Amicon), and mtSSB was purified to apparent
homogeneity by preparative electrophoresis on a 15% sodium dodecyl
sulfate-polyacrylamide gel using the Prep-Cell (Bio-Rad). Purified
antigen (100 µg) was mixed with an equal volume of Titer-Max adjuvant
(CytRx Corp.) and injected both intramuscularly and subcutaneously into
a New Zealand White rabbit. Blood and serum samples were acquired by standard methods. The antiserum was purified further by precipitation in 33% ammonium sulfate.
Cell Culture and Histochemistry-- Murine C2C12 myogenic cells were propogated as described previously (15) and plated onto gelatin-coated coverslips and further cultured for 24 h. Cells attached to coverslips were rinsed in cold PBS fixed with 4% paraformaldehyde for 10 min at 4 °C, rinsed, permeabilized with 0.1% Triton X-100, and incubated in block solution (1.0% bovine serum albumin) for 10 min. Cells were hydrated in PBS, incubated with 10% normal goat serum to reduce background staining for 30 min at room temperature and incubated overnight at 4 °C with primary antibody (anti-mtSSB). On the following day, the cells were washed, incubated with the secondary antibody (fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson Immunoresearch)) and Mitotracker dye (Molecular Probes), washed further, and mounted on glass slides with Vectashield mounting media (Vector Laboratories). Substitution controls with either normal rabbit serum or PBS were performed, and in all instances the controls were negative.
Human lymphoblastoid cell lines were grown in RPMI medium and fibroblasts in Dulbecco's modified Eagle's medium, each supplemented with 15% fetal bovine serum and 4 mM L-glutamine. Cell lines included cytogenetically normal lymphoblastoid (SC-1) and fibroblast (SMITO-1 and GM01604) controls, as well as a line designated SA15q-3 derived from a patient diagnosed with a direct duplication of the distal portion of chromosome 15 (dir dup(15)(pter-q24.1::q15-q26.2::q25.2-qter)). This structural rearrangement was expected to result in triploidy for the PolQuantitation of Results and Statistical Analysis-- Autoradiographs of Northern blots were optically scanned to generate a quantitative estimation of mRNA abundance relative to 28 S rRNA or to GAPDH within each RNA extract. Data from Southern blots were quantified in a similar manner. In the experiments involving motor nerve stimulation of rabbit skeletal muscles or post-natal development of the mouse heart, three-six animals were examined at each time point. Differences between group means were assessed using Dunnet's post-hoc test with significance defined as p < 0.05.
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RESULTS |
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Expression of POLCAT mRNA in Different Mammalian
Tissues--
Mammalian tissues differ markedly with respect to the
abundance of mtDNA (2) and to expression of certain nuclear genes encoding mitochondrial proteins (4, 17). Northern analysis indicated
that expression of Pol
CAT mRNA in a variety of human tissues bears no apparent relationship to mitochondrial mass or mtDNA
content. After normalization to an internal standard (GAPDH mRNA),
expression of Pol
CAT mRNA in heart differed by no
more than 2-fold from levels observed in any other human tissue
(pancreas, kidney, liver, lung, brain, colon, prostate, thymus, and
spleen; data not shown). The expression of Pol
CAT
mRNA in mitochondria-rich cardiac tissue by comparison to lung is
illustrated in Fig. 1A where
the quantitative analysis revealed expression in the lung to be
approximately 2-fold greater than that found in the heart. In rabbit TA
muscles subjected to chronic stimulation via the motor nerve, a potent
stimulus to mitochodrial biogenesis and mtDNA replication (3), there
was no detectable induction of expression of POL
mRNA (Fig.
1B). The magnitude of the increase in mtDNA in this set of
animals (3.5-fold at 10-21 days) was comparable with that observed
previously in our laboratory (3, 13).
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Expression of POLCAT mRNA Is Influenced by Gene
Dosage Without Changes in mtDNA--
A Northern blot was performed
with mRNA from normal control cell lines (SC-1, SMITO-1, and
GM01604) and a line established from a patient expected to be triploid
for the Pol
CAT locus (SA15q-3). Expression of POL
mRNA was elevated approximately 3-fold over that seen in the
diploid control cell lines (Fig.
2A). Southern blots of DNA
derived from control and SA15q-3 cell lines were probed with a fragment
of the Pol
CAT gene or with a sequence (D15S10) mapping
to the proximal non-duplicated region of the chromosome. The results
demonstrated a 9-kb HindIII Pol
CAT band consistent with a 9,081-base pair size predicted from genomic sequencing of this region.2 Moreover, the results confirm
that the Pol
CAT locus is triploid in this cell line
(Fig. 2B). Quantitation of the relative band intensities
revealed an SA15q-3:control ratio for Pol
CAT of 1.52 when normalized for the diploid marker D15S10. Southern blots probed
with a fragment of human mtDNA encoding the cytochrome b
gene demonstrated a 10-kb band, consistent with a size of 10,202 base
pairs predicted from the human mtDNA sequence (18). A quantitative comparison of the relative intensities of the mtDNA hybridization signals from SA15q-3 and control cells (Fig. 2B) was
calculated at a ratio of 1.06, demonstrating that the increased
expression of Pol
CAT mRNA is not associated with a
greater abundance of mtDNA in this cell line in which the
Pol
CAT locus is amplified.
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Regulated Expression of mtSSB mRNA in Mammalian
Tissues--
Using rabbit mtSSB cDNA as the probe under conditions
of high stringency, two transcripts with molecular weights of
approximately 940 and 650 bases were detected in RNA extracted from
rabbit muscles (Fig. 3, A and
B). The smaller of the two transcripts corresponds to the
size of mtSSB mRNA described previously in rat and human cells (9)
and to the single transcript observed in murine cells (Fig.
3C). Drosophila express two transcripts of 0.6 and 1.5 kb that hybridize to a mtSSB cDNA probe (19), and two
isoforms of mtSSB have been described in Xenopus laevis
(20), but this is the first demonstration of multiple mtSSB transcipts
in a mammalian species. The relative abundance of the 650 base
transcript correlated directly with the oxidative capacity, fractional
volume of mitochondria, and mtDNA content (2) of individual
striated muscles of this species (heart soleus > plantaris > gastrocnemius > EDL = TA) (Fig.
3A).
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Subcellular Localization of mtSSB-- In mammalian cells, replication of mtDNA takes place preferentially within a subset of mitochondria clustered within a perinuclear domain (12). We observed that mtSSB protein is not distributed uniformly within all mitochondria of murine C2C12 myogenic cells but is more abundant within perinuclear regions (Fig. 4). Mitochondria within cell processes more distant from the nucleus are devoid of mtSSB, at least at the level of detection of this immunofluorescent technique.
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DISCUSSION |
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The principal observation of this study is that expression of
nuclear genes encoding mitochondrial DNA replication factors in
mammalian cells is regulated in a differential rather than a
coordinated manner. Expression of mRNA encoding the catalytic subunit of POL is constitutive among specialized tissues that differ
markedly in their relative contents of mtDNA. Likewise, we could detect
no appreciable up-regulation of POL
CAT mRNA in skeletal muscles subjected to chronic motor nerve stimulation, a potent
stimulus to mtDNA replication. Other investigators have noted
constitutive expression of POL
CAT mRNA and protein
in human tumor cells depleted of mtDNA by exposure to ethidium bromide as compared with the parental cell line (22). Mammalian cells, therefore, appear to lack a feedback mechanism to respond to a deficiency of mtDNA by up-regulation of POL
CAT gene
expression.
In contrast, expression of mtSSB mRNA correlates directly with the
abundance of mtDNA within specialized muscle subtypes. This
relationship is consistent in three independent analyses: (a) the comparison of different striated muscles within
adult animals, (b) the response of the rabbit tibialis
anterior skeletal muscle to chronic motor nerve stimulation, and
(c) post-natal development of the murine heart. We conclude
that expression of the mtSSB gene, unlike the POLCAT
gene, is regulated by developmental cues or physiological stimuli that
promote changes in mitochondrial biogenesis. Preliminary immunoblot
analyses3 indicate that mitochondria-rich striated muscles
contain greater amounts of mtSSB protein, corresponding to the changes
in mtSSB mRNA described in this paper. Moreover, we demonstrate
here that mtSSB is preferentially localized to a perinuclear subset of
mitochondria that are active sites for mtDNA replication (12).
How should these findings be interpreted with respect to the
biochemical and molecular mechanisms that govern replication and
maintenance of mtDNA in mammalian cells? Constitutive expression of the
POLCAT gene in tissues with differing requirements for mtDNA replication suggests that, under physiological conditions, the
enzymatic activity of POL
is not constrained by limited expression of this subunit. Indeed, in a previous study we observed an increased specific activity of POL
in mitochondria-rich muscle tissues (21) in
comparison with glycolytic muscles that we now show to express a
comparable abundance of POL
CAT mRNA. We cannot
exclude the possibility that the abundance of the catalytic subunit of POL
is regulated independently of changes in its mRNA through mechanisms that modify translational efficiency or stability of the
protein. A more likely explanation, however, is that the availability of other mtDNA replication factors, and not POL
CAT, is
rate-limiting to mtDNA replication. Our observation that the cellular
content of mtDNA is not increased when the POL
CAT gene
is amplified and overexpressed supports this viewpoint.
The abundance of mtSSB could influence the cellular content of mtDNA in
one of several ways. Since the activity of purified POL is
stimulated by mtSSB in vitro (10, 11), it is possible that
expression of mtSSB likewise is rate-limiting to mtDNA replication in vivo. Alternatively, higher concentrations of mtSSB may
increase the stability of mtDNA, particularly under conditions where
partially replicated D-loop forms of the mitochondrial genome are
abundant (21). The consistent correlation between expression of the
mtSSB gene and the cellular content of mtDNA among different mammalian tissues supports the notion that increased intramitochondrial concentrations of mtSSB are required for, even if not the primary regulatory determinant of, the greater abundance of mtDNA in cells faced with high physiological demands for mitochondrial
respiration.
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ACKNOWLEDGEMENTS |
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We thank Maggy Fina and John Shelton for valuable technical contributions. We are also grateful to Drs. Massimo Zeviani and Stephen Johnston for plasmids.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grants HL06296, HL54794, and CA52121 and by National Institutes of Health Training Grant HL07360 (to S. J. S. and E. C. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
Texas Southwestern Medical Center, NB11.200, Dallas, TX 75235-8573. Tel.: 214-648-1400; Fax: 214-648-1450; E-mail:
williams{at}ryburn.swmed.edu.
1
The abbreviations used are: mtDNA, mitochondrial
DNA; POL, DNA polymerase
; POL
CAT, catalytic
subunit of DNA polymerase
; mtSSB, mitochondrial single-stranded
DNA-binding; PCR, polymerase chain reaction; kb, kilobases; TA,
tibialis anterior; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
GST, glutathione S-transferase; PBS, phosphate-buffered saline.
2 L. D. McDaniel and R. A. Schultz, unpublished observation.
3 S. Swoap and R. S. Williams, unpublished results.
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REFERENCES |
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