Regulation of skeletal a-actin promoter in young
chickens during hypertrophy
caused by stretch overload
JAMES A. CARSON, ZHEN YAN, FRANK W. BOOTH, MICHAEL
E. COLEMAN,
ROBERT J. SCHWARTZ, AND CRAIG S. STUMP
Department of Physiology and Cell Biology, University of Texas-Houston Health Science Center
and Department
of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
Carson,
James A., Zhen Yan, Frank
W. Booth,
Michael E. Coleman,
Robert
J. Schwartz,
and Craig
S.
Stump.
Regulation
of skeletal a-actin promoter
in young
chickens during hypertrophy
caused by stretch overload. Am.
J. Physiol.
268 (CelZ PhysioZ. 37): C918-C924,
1995.Anterior
latissimus
dorsi (ALD) muscles of 3-wk-old
male
chickens were injected with plasmids containing various lengths
of the chicken skeletal cx-actin promoter (ranging from -2,090
to -77 relative to the transcription
start site) driving luciferase. Hypertrophy
of the left ALD muscle was induced by
attaching
a weight (11% of body wt) to the left wing of each
chicken, with the unweighted
contralateral
wing serving the
control. Six days of stretch overload significantly
increased
muscle mass 110%. Luciferase
activity
from the -2,090
actin-luciferase
chimeric gene increased 127% compared with
the contralateral
control ALD muscle. Luciferase
activities
driven by the -424, -202, and -99 actin promoters were 179,
134, and 378% higher,
respectively,
in the stretched
ALD
muscle than in the contralateral
control ALD muscle. Luciferase activity from the -77 deletion construct was not different
between stretched and control muscles. These data indicate
that the gene region responding
to stretch is downstream
of
-99 and imply, but do not conclusively
prove, that the region
between -99 and - 77, which contains serum response element 1, contributes
to the stretch-induced
increase in skeletal
at-actin promoter activity in the ALD muscle.
muscle; exercise; contractile
protein;
gene
of skeletal muscle occurs in response to
overload, but little is known about which regulatory
sequences of genes are activated to cause the increased
accumulation
of contractile tissue in living animals (6).
Stretch overloading of the chicken anterior latissimus
dorsi (ALD) muscle results in an 80% increase in protein
content after 7 days of chronic stretch overload (16) and
a 140-225%
increase in the synthesis rate of actin
protein at l-3 days of stretch overload (11). These data
demonstrate
that a-actin gene expression is increased
during stretch-induced
hypertrophy.
Regulation of expression level, tissue specificity, and
development
are mediated by sequences within the
region -2,000 to +239 of the transcription
start site of
the human skeletal a-actin gene in transgenic mice (7).
The region -153 to -87 base pairs (bp) from the
transcription
start site of the human skeletal oc-actin
promoter is both sufficient and necessary for musclespecific expression and developmental regulation during
myogenesis (7). Chicken skeletal ar-actin gene regulation
in primary myogenic culture has been well described (4,
19). The first - 200 bp 5’ of the chicken skeletal oc-actin
transcription
start site give tissue-specific regulation of
the gene in myogenic cell culture (4) and transgenic mice
ENLARGEMENT
C918
0363-6143/95
$3.00 Copyright
(28). This region contains consensus cis-acting DNA
regulatory sequences that include three serum response
elements (SRE)/CArG/CBAR,
E box, transcription
enhancing factor l/M-CAT binding site, two Spl sites, and
TATA box (4). These cis-acting elements and their
orientation
in the proximal promoter
region of the
skeletal ar-actin gene have been strongly conserved
during evolution in chickens, mice, rats, and humans
(23, 26). The chicken and rat skeletal ar-actin genes
contain four highly conserved regions found between
-80 and -230 bp from the transcription
start site (25).
These include regions corresponding to cis-acting DNA
regulatory elements SREl and SRE2 and an E box,
which binds the MyoD family of myogenic proteins. A
consensus transcription-enhancing
factor 1 binding site
(-66 to -60) is also conserved between the rat and
chicken skeletal a-actin promoters
(25). Despite this
large amount of information,
less is known about the
activity of the chicken skeletal a-actin promoter during
skeletal muscle hypertrophy
in living animals.
Plasmid DNA injected into skeletal muscle has been
shown to be taken up by muscle fibers, to be stably
expressed, to produce proteins for antibody production
by the injected muscle, and to be useful for promoter
analysis (1, 8, 20, 29, 36, 37, 39-41). We wanted to test
whether direct plasmid injection methodology
would
allow the study of skeletal ar-actin gene regulation using
an established animal model to elicit hypertrophy. Deletions of the 5’-flanking region of the chicken skeletal
ar-actin gene driving expression of the firefly luciferase
coding region were injected into stretch-overloaded
and
contralateral
control ALD muscles of rapidly growing
chickens. The purposes of the present study were to
determine whether chimeric gene expression from the
-2,090-bp
ar-actin promoter is increased during hypertrophy of the ALD and, if there is an increase in
expression, to define a region within -2,090 bp of the
5’-flanking region of the skeletal cx-actin promoter that
may have elements responding to stretch overload.
MATERIALS
AND METHODS
Animal
care and preparation.
One-day-old
roosters (White
Leghorn
males, Ideal-286,
Ideal Hatcheries,
Cameron,
TX)
were received and housed at the animal care facilities, University of Texas Health Science Center at Houston. The chickens
were provided chicken laboratory
diet and water ad libitum in
a chicken brooder with a 12:12-h light-dark
cycle. At - 3 wk of
age the chickens were housed two per cage for the duration of
the study. The chickens used in the study were between 3 and
5 wk of age (200-400 g). All animal protocols were approved by
the Institutional
Animal
Welfare Committee,
University
of
Texas Health Science Center.
o 1995 the American
Physiological
Society
ACTIN
PROMOTER
IN
MUSCLE
PLasmids. A 2.3-kb fragment
containing
2,090 bp of the
chicken a-actin promoter
(4) was subcloned into pGL2 basic
plasmid (Promega) containing
the cDNA for luciferase (Fig. 1).
Briefly, the 2.3-kb actin insert was removed from its pBR322
vectors with restriction
endonuclease
Hind III, purified
by
electrophoresis,
and subcloned into the Hind III site of pGL2
basic. pGL2 basic, without
the actin insert, was injected for
promoterless
luciferase expression.
A 448bp
fragment
of the chicken skeletal cx-actin gene
containing
424 bp 5’ of the transcription
start site (4) was
subcloned into pGL2 basic plasmid containing
the cDNA for
luciferase. Briefly, the 448bp actin insert was removed from
its pTZ19R vector (4) with restriction
endonuclease
Hind III,
purified by electrophoresis,
and subcloned into the Hind III
site (+47) of pGL2 basic. The -77-bp cx-actin promoter
was
made by cutting the -448 a-actin insert with restriction
endonuclease
EcZ XI (-74) and cutting the multiple
cloning
region of pGL2 basic with restriction
endonuclease
Xho I
(+33), blunt ing the sticky ends with the Klenow fragment, and
religating
the plasmid.
The -99-bp
a-actin promoter
was
made by cutting the -424 a-actin insert with restriction
endonuclease
Apa I (-98) and the multiple
cloning region of
pGL2 basic with Sma I (+ l), blunting
the sticky ends created
by Apa I with the Klenow
fragment,
and religating
the
plasmid. The -202-bp
cx-actin promoter
was made by cutting
the -424 a-actin insert with restriction
endonuclease
Sma I
(-202) and th e multiple
cloning region of pGL2 basic with
Sma I (+ 1) and religating
the plasmid. All a-actin constructs
sequences and were verified
by DNA sequencing
(United
States Biochemical)
(Fig. 1).
A plasmid containing
the long terminal
repeat of the Rous
sarcoma virus (RSV) directing the cDNA for chloramphenicol
acetyltransferase
(CAT) was also used in the study. The
plasmid pRSVCAT
contains RSVCAT
in vector pBR322, a
low-copy-number
vector. The plasmid yield was improved by
subcloning the RSV promoter and CAT coding region from the
pRSVCAT plasmid into the high-copy-number
vector pUC18.
pOCAT was injected to obtain promoterless
CAT expression.
PLasmid purification.
Plasmid DNA was transformed
into
XLl-Blue
bacteria (Stratagene).
The bacteria were then grown
for 15 h in LB medium
with moderate
shaking at 37°C.
Plasmids were isolated and purified using alkaline lysis with
differential
polyethylene
glycol precipitation
and subsequent
phenol extractions
(31). DNA concentration
and purity was
determined
by ultraviolet
spectrophotometry
using AzGO and
A260-to-A280 ratio. Plasmid preparations
were then subjected to
analysis by restriction
endonuclease
digestion and agarose gel
electrophoresis
to demonstrate
the DNA was correct and free
SRE3
-2090
-2090
SRE2
SREl
TEFl
-185 -130
-85
-65
I-'/
from contamination.
Plasmid DNA was stored in sterile dHzO
at -80°C until the time of use.
Directplasmid
injection. The right and left ALD muscles of
3-wk-old
chickens were injected
with plasmid
DNA using
methodology
described by Wolff et al. (41). Chickens were
anesthetized
using a ketamine-xylazine-acepromazine
(25, 1,
and 1.5 mg/kg, respectively)
cocktail. A single incision was
made along the spine adjacent to the right and left ALD
muscles. The ALD muscles were visualized
and preinjected
with 25 ~1 of sterile 25% sucrose (8) at the midbelly
of the
muscle with a 27-gauge needle. Approximately
20 min later 50
pg of the appropriate
actin-luciferase
plasmid and 50 kg of
RSVCAT plasmid were injected together in a final volume of 20
~1. A single-track
injection of plasmid DNA at the midbelly of
the muscle was performed
over a 30-s time period using a 25-~1
Hamilton
syringe with a sterile 27-gauge needle. The -2,090
actin-luciferase
DNA (65 pg) was injected with 50 pg of
RSVCAT to make the DNA injected equal in molar amount to
the smaller length promoter constructs. Interestingly,
similar
methodology
used to inject plasmid DNA into the patagialis
muscle of the chicken did not illicit consistent expression.
Wing weighting.
Two days after having been injected with
the appropriate
plasmid
DNA, the chickens had a weight
corresponding
to 11% of their body weight attached to the left
wing. The contralateral
wing was not weighted and served as
the control
(2). After 6 days of wing weighting
the ALD
muscles were removed, frozen in liquid N2, and stored at
-80°C.
Assays for Zuciferase and CAT activity. ALD muscles were
homogenized
in l-3 ml of homogenate
buffer [25 mM tris(hydroxymethyl)aminomethane
(Tris), 5.7 mM trans-1,2-diaminocyclohexane-N,N,N’,N’-tetraacteic
acid, 10% glycerol, 2 mM
dithiothreitol
(DTT), 1 mM phenylmethylsulfonyl
fluoride, 1
mM EDTA, 1 mM bezamidine,
0.01 mg/ml leupeptin, and 0.01
mg/ml pepstatin]
using a Polytron homogenizer
(Kinematica,
Switzerland)
at 60% of maximum
setting (3 x 10 s) and then
centrifuged
(10,000 g, 10 min, 4°C). The supernatant
containing the crude protein extract was analyzed for luciferase and
CAT activity. Luciferase activity was determined
using 20 ~1 of
muscle extract incubated
for 70 s with 100 ~1 of luciferase
reagent produced by mixing 20 mM tricine (pH 7.8), 1.07 mM
(MgCO&Mg(OH)2*5H20,
2.67 mM MgS04, 0.1 mM EDTA,
0.53 mM ATP, 0.27 mM coenzyme A, 33.3 mM DTT, and 0.47
PM luciferin.
Light production,
quantified
as relative light
units (RLU), was measured
by a luminometer
(Analytical
Luminescence
Laboratory
Monolight
2010) for 10 s and
integrated
over time. The RLU for each sample was corrected
for differences in ALD muscle mass by calculating
the total
+l
+24
+318
/m
-424
i
-99
i-1
-77 1-i
c919
HYPERTROPHY
Fig. 1. Conserved
regulatory
elements
of chicken
skeletal
cx-actin gene including serum
response
elements
(SREl,
SRE2,
and SRE3)
and transcriptionenhancing
factor
1 (TEF-1)
binding
site.
Five actin promoter
deletions
used were
from -2,090
(n = 17 birds),
-424
(n =
18 birds),
-202 (n = 9 birds),
-99 (n =
15 birds),
and -77
(n = 20 birds)
bp
from
transcription
start
site.
-424,
-202,
-98, and -77 all had same 3’ end
relative
to transcription
start site (+24).
-2,090
promoter
3’ end was at +318
from transcription
start site.
c920
ACTIN
PROMOTER
IN
luciferase activity for the volume of each muscle homogenate
(TRLU).
CAT activity was assayed using [ l*C]chloramphenicol
(Amersham Life Sciences) as the substrate (31). Muscle homogenate (50 ~1) was incubated
(37”C, 2 h) with 130 ~1 of assay
buffer 1192 mM Tris (pH 7.8), 1.8 mM acetyl-CoA, and 0.2 PCi
[ 14C]chloramphenicol].
The acetylated
chloramphenicol
was
separated using thin-layer
chromatography
(30 min, 20°C) in a
tank containing
95% chloroform
and 5% methanol and visualized by autoradiography.
The portions of the plates containing
the acetylated
and unacetylated
chloramphenicol
for each
sample were excised and quantified
by scintillation
counting.
The percentage conversion of acetylated chloramphenicol
was
presented as the percentage conversion per minute. The CAT
activity was corrected for the volume of muscle homogenate
to
give percentage conversion per minute per muscle. This value
was then used to correct TRLU activity in TRLUICAT
for
each muscle (37). Only birds with CAT activities
>0.25
%conversion
min-l *muscle-l
in both the control
and the
stretched ALD were utilized
in the study (15). Uninjected
muscles were analyzed each day of assays, and they served as
the background,
which was subtracted
from samples run on
that day.
For each muscle, the ratio of the two reporter
genes was
calculated: luciferase activity/CAT
activity = TRLU/fraction
of chloramphenicol
that is acetylated. Values of luciferase/
CAT for each muscle are used for calculation
of means 2 SE in
each treatment group.
MyobZast cuZture and transfection.
Primary
embryonic
chicken myoblast cultures were established from breast tissue
of 1 l-day-old
chicken embryos as described previously
(13).
Myoblasts
were plated at a density of 8 x lo5 cells into
collagen-coated
60-mm plastic Petri dishes in minimum
essential medium supplemented
with 10% horse serum, 5% chicken
embryo extract, and 50 kg/ml gentamicin
sulfate and maintained at 37°C and 5% CO2 in a humidified
incubator.
Myoblasts were transfected 24 h postplating
with 5 pg/plate of the
same chicken skeletal cx-actin promoter
constructs
that had
been injected into the ALD muscle and 2 pg/plate pCMVGAL
(Clonetech, Palo Alto, CA) using DEAE-dextran
essentially as
described
previously
(22). Briefly,
myoblasts
were washed
three times with Hanks’ balanced salt solution,
3 ml/plate
minimum
essential medium containing
specified DNA plasmid
constructions
and 150 pg/ml DEAE-dextran
(Sigma, St. Louis,
MO) were then added, and myoblasts were incubated for 3 h at
37°C in a humidified
incubator.
After 3 h, media were aspirated and myoblasts were exposed to a 10% dimethyl sulfoxide
(DMSO) shock (90% minimum
essential medium
and 10%
DMSO) for 90 s. Myoblasts were then washed three times with
Hanks’ balanced salt solution, fed with 3 ml/plate
minimum
essential medium supplemented
with 10% horse serum, 2%
chicken embryo extract, and 50 kg/ml gentamicin
sulfate, and
maintained
at 37°C and 5% CO2 in a humidified
incubator.
After 45 h (i.e., N 72 h postplating),
myoblasts/myotubes
were
washed three times with phosphate-buffered
saline and then
lysed in 200 kl/plate 0.25 M Tris Cl (pH 7.8) and 0.1% Triton
X-100. Myotube lysates were transferred
to 1.5-ml microcentrifuge tubes and then clarified by centrifugation
at 12,000 g for
5 min at 4°C. The supernatants
were stored at -70°C for
subsequent luciferase and P-galactosidase
(3 1) assays.
RNA isolation.
Stretched
and control ALD muscles were
divided into two pieces, a 20- to 40-mg piece of muscle was
saved at -80°C for total RNA determination
(see below), and
the remainder
was powdered in liquid N2 for cx-actin mRNA
determination.
RNA was extracted using the RNAzol B method
(Biotecx Laboratories,
Houston,
TX). Briefly, muscle tissue
was homogenized
with RNAzol B and treated with chloroform
l
MUSCLE
HYPERTROPHY
(0.1 vol) to remove proteins and DNA. RNA was precipitated
with isopropanol
(1 vol), and the RNA pellet was dissolved in
diethyl pyrocarbonate-treated
dH20. RNA concentration
and
purity were determined
by ultraviolet
spectrophotometry
at
A260
and
A2809
a-Actin mRNA determination.
Northern
blot analysis was
used to quantify
the abundance
of a-actin mRNA in the
control and stretched
ALD muscles. Isolated RNA (10 pg)
from each muscle was loaded onto a denaturing
1% agarose gel
[ 1 x 3-(N-morpholino)propanesulfonic
acid and 6.7% formaldehyde] and electrophoresed
at 5 V/cm for 2.5 h. The RNA was
then transferred
to a nylon membrane
by capillary
action.
Upon completion
of the transfer the RNA was ultraviolet
cross
linked to the membrane.
A chicken skeletal <x-actin cDNA probe was prepared by
random
priming
(9) of a unique
221-bp fragment
of the
chicken skeletal a-actin cDNA taken from the 3’-untranslated
region (UTR) of the chicken a-actin gene. DNA template for
labeling was prepared by polymerase chain reaction amplification of the unique 3’-UTR fragment from the clone (x-SK.22
described previously
( 13).
The membrane containing
the RNA was prehybridized
with
12-ml hybridization
buffer (QuickHyb,
Stratagene)
for 30 min
at 68OC. An aliquot of cx-actin mRNA probe (3.7 x lo7 cpm; sp
act log cpm/pg DNA) was mixed with the hybridization
buffer
and incubated for 2 h at 68°C. The membrane was washed two
times with 2 x sodium chloride-sodium
citrate (SSC)-0.1%
sodium dodecyl sulfate (SDS) (2O”C, 15 min) and once with
0.1 x SSC-0.1% SDS (55”C, 30 min). The membrane was then
visualized
by autoradiography
(-8O”C,
3 h), and the band
corresponding
to a-actin mRNA was then quantified
by densitometry scanning (Bio Image, Millipore,
Ann Arbor, MI) as the
integrated
optical density (IOD) and used to calculate IOD
cx-actin mRNA per microgram
of RNA. The quantity of cx-actin
mRNA was normalized
to ethidium bromide-stained
28s RNA,
which was determined
by image analysis (Bio Image) (5).
TotaZ RNA determination.
Total RNA was quantified
using
the method of Fleck and Munro (10).
Statistics. The data are expressed as means * SE. A paired
t-test was used to determine differences between the stretched
and control ALD, and P < 0.05 was designated significant.
RESULTS
Body weight and ALD mass. The body weights of the
chickens (n = 69) were 202.9 t 3.2 g at the time of
injection (day O), 211.9 t 3.8 g at the time of wing
weighting (day Z), and 283.5 t 5.8 g after 6 days of wing
weighting
(day 8). The wet weight of the stretchoverloaded ALD muscle (226.8 t 4.8 mg) was increased
significantly
(110%; n = 69, P = 0.0001) above the
contralateral
control ALD (107.9 t 2.7 mg) after 6 days
of wing weighting.
Total RNA and cu-actin mRNA. Six days of wing
weighting caused the total RNA per gram of wet weight
to increase significantly (42%; n = 9, P = 0.003) in the
stretched ALD muscle (3.00 t 0.09 mg RNA/g muscle)
compared with the contralateral control (2.11 t 0.11 mg
RNA/g muscle). Total RNA for the entire muscle in the
stretch-overloaded
ALD was 239% greater than in the
contralateral
control ALD (755.0 kg RNA/muscle
vs.
223.5 pg RNA/muscle, respectively).
cx-Actin mRNA per microgram of RNA was decreased
significantly
(32%; n = 9, P = 0.01) in the stretchoverloaded ALD compared with the control muscle
(Table 1, Fig. 2). Th is was due to the increase in total
ACTIN
PROMOTER
IN
MUSCLE
c921
HYPERTROPHY
Table 1. Skeletal a-actin mRNA
Control
IOD actin
IOD actin
IOD actin
mRNA/pg
RNA
mRNA/g
muscle
mRNA/whole
muscle
Values are means
? SE. Skeletal
RNA,
from
control
and stretched
integrated
optical
density
(see Fig.
control.
0.107 * 0.015
0.22520.034
0.0249 2 0.0050
Stretch
0.072 + 0.018*
0.197 % 0.035
0.0492?
0.0091*
a-actin
mRNA,
corrected
for 28s
ALD muscle
(n =9 birds).
IOD,
2). *P < 0.05 from contralateral
F
3
a 1500w-
- wwooo
I-
lOOOOO-
-4000000
5aOQO-
RNA concentration. There was no significant difference
(P = 0.27) between the control and stretch-overloaded
ALD in the actin mRNA per gram of wet weight (Table
1). The quantity of cY-actin mRNA in the whole ALD
muscle was increased significantly (98%, P = 0.002) by
stretch overload above the contralateral control (Table
1). Plasmid injections had no effect on quantity of actin
mRNA in the stretched or control ALD muscles (data
not shown).
Dose-response curve for chimeric gene expression from
-424-bp a-a&in promoter. A dose-response curve was
done with varying quantities (25-100 kg) of the -424-bp
a-actin promoter directing the cDNA for luciferase
coinjected with 50 Fg of RSVCAT plasmid (Fig. 3). A
50-kg dosage of the -424 actin/luciferase
construct was
in the linear range of chimeric gene expression when
examined for total luciferase activity and luciferase
activity corrected for CAT activity.
Promoter-less expression. Luciferase activity determined from ALD muscles injected with 50 kg of pGL2
basic was 1,537 + 787 in untreated wings and 1,730 +
783 in stretched wmgs (n = 13; Table 2). These activities
are 10 and 16% of those found in control and stretch
muscles, respectively, receiving 50 p,g of -77 actin
directing luciferase. CAT activities were not detectable
in muscles injected with 50 kg of a promoterless CAT
construct (pOCAT).
Chicken a-a&in promoter activity. The longest length
of the chicken o-actin promoter in this experiment was
-2,090 bp from the a-actin transcription
start site. The
C
3
3
-2oooooO
~
04 =
20
40
424
60
actbl/luoiferase
00
loo
DNA
IO
120
(Jig)
Fig. 3. Dose-response
relationship
of chimeric
gene expression
in
unweighted
ALD muscle.
ALD muscles
were injected with 25 (n = lo),
50 (n = 12), 75 (n = 9), and 100 kg (n = 10) of -424-bp
a-actin
promoter
and 50 kg of Rous sarcoma
virus chloramphenicol
acetyltransferase
(pRSVCAT).
Expression
was measured
after
8 days.
Expression
is expressed
as total
relative
light
units
(TRLU,
l )
corrected
for CAT activity
(: I).
left and right ALD muscles were injected with 65 kg of
-2,090 actin-luciferase plasmid and 50 pg of RSVCAT,
and the left wing weighted 2 days postinjection. Activity
from the -2,090-bp promoter, corrected for RSVCAT
expression, was significantly increased by 127% (P =
0.05; n = 17) in the hypertrophied
ALD compared with
the contralateral control during 6 days of stretch overload (Fig 4). This increase in luciferase/CAT
expression
was due to a greater increase in TRLU than the increase
in CAT activity in the hypertrophied muscle (see Table 2).
Deletion analysis was performed on the a-actin promoter to determine the region responsible for the increase in chimeric gene expression during stretchinduced hypertrophy.
Four deletions of the -2,090 bp
promotor were used (Fig. 1). The appropriate deletion
construct (50 kg) was coinjected with 50 p,g of RSVCAT
into the right and left ALD. o-Actin promoter activity in
the control ALD muscle increased as the a-actin promoter length increased, peaking with the -424-bp
promoter and decreasing at -2,090 bp (Fig. 4).
a-Actin chimeric gene expression, corrected for RSVCAT expression, was elevated significantly in the hypertrophied ALD compared with the contralateral control
in birds injected with plasmid DNA containing -424
(179%, P = 0.004), -202 (134%, P = 0.0184), and -99
(378%, P = 0.0007) promoters (Fig. 4). The corrected
chimeric gene expression from the -77-bp promoter
was not altered significantly (P = 0.2891) in the hypertrophied ALD (luciferase/CAT
= 74,310) compared with
the contralateral
control (luciferase/CAT
= 495,610;
Fig. 4). These data demonstrate that the 22-bp region
located from -77 to -99 bp from the a-actin transcription start site could contain a regulatory element(s)
responsible for the increase in chimeric gene expression
during stretch overload-induced hypertrophy.
Expression of skeletal a-actin promoter in cultured
muscle cells. Progressive deletions in the first 200 bp
Fig. 2. Northern
analysis
of o-actin
mRNA
from control
stretched
(S) anterior
Iatissimus
dorsi (ALD)
muscle.
Total
kg) was added to each lane. See Table 1 for tabulated
results.
(0
RNA
and
(10
(- 77, - 99, and - 202) of the cu-actin promoter produced
a pattern of activity in control ALD (Fig. 4) and cultured
chicken muscle cells (Fig. 5), which closely matched the
c922
ACTIN
PROMOTER
IN MUSCLE
HYPERTROPHY
Table 2. Reporter gene expression
TRLU
CAT
Plasmid
n
Control
Stretch
1,537*787
pGL2 basic and RSVCAT
pOCAT and RSVLUC
pActLUC
and RSVCAT
2,396,160
-77
-99
-202
-424
1,730&783
5,234,785+2,021,451
2 1,072,892
15,637+5,777
13,305+5,817
26,632k 13,719
454,675+ 196,011
-2090
Stretch
8.572 1.79
0.00 2 0.00
11.64k2.83
0.01~0.00
13
18
10,539*4,534
8.15 2 1.57
63,013*16,577*
8.6521.82
6.8722.41
14.64t2.25*
13.90 + 2.29
15
109,978 + 42,242”
1,083,947
+ 239,316”
56,969+18,353*
11,148*1,857
Control
12.01+3.20*
7.49 2 1.35
3.4120.63
10.612
2.09
7.4222.27
20
9
18
17
Values are means + SE. Each plasmid
(50 pg) was coinjected
into the anterior
latissimus
dorsi (ALD) muscles
of chickens.
TRLU,
total relative
light units
per muscle
for luciferase
expression;
CAT,
enzymatic
activity
of chloramphenicol
acetyltransferase
(CAT)
given as percent
conversion
of acetylated
chloramphenicol;
Control,
group with contralateral
nonstretched
right ALD muscle;
Stretch,
group with ALD muscle in
left wing that had been weighted
with 11% of body weight;
pGL2 basic, promoterless
luciferase;
pOCAT,
promoterless
CAT; RSV, Rous sarcoma
virus promoter
driving
either luciferase
(LUC) or CAT. Negative
numbers
under pActLUC
promoter
indicate
promoter
length.
*P < 0.05 from
contralateral
control.
expression pattern
previously
reported
in cultured
chicken myocytes (4). However, it has previously been
reported that the - 2,000, -422, and -200 length chicken
skeletal cx-actin promoter activities were not different
when driving the reporter gene CAT (4). We have found
that the activities of these promoters driving the reporter gene luciferase in control muscles of the live
animal and in cultured myocytes are different, with the
- 424 promoter
expressing significantly
higher than
either the -202 or -2,090 cx-actin promoter constructs
(Figs. 4 and 5). W e speculate that either the shorter
half-life of luciferase relative to CAT or the method of
transfection could account for these differences (32).
DISCUSSION
Results of the current study suggest that a region
which is within the first 99 bp of the chicken ar-actin
promoter is required for the stretch-induced
increase in
the activity of this promoter. Furthermore,
these data
13000000
12000000
1
0 Control
W Stretch
suggest that the region between - 77 and -99 may
contain DNA regulatory
elements necessary for this
stretch-induced
increase in expression. This region corresponds to the location of a cis-acting DNA regulatory
element called SREl, consisting of the sequence 5’CCAAATATGGC-3’
from - 81 to - 91 bp. SRE 1 matches
the consensus sequence reported for the SRE (5’-CC(A/
T),GG-3’) (34) and is the most proximal of three serum
response elements found in the chicken skeletal a-actin
promoter (5).
The consensus motif for the SRE has been found in
the 5’-flanking regions of many growth factor-inducible
genes (34) and muscle-specific genes (19, 21, 27). However, the SRE was first described in the regulation of the
c-fos protooncogene
(34). Increased c-fos transcription
in cultured cells after serum induction was found to be
mediated through the SRE in the c-fos promoter (33).
Subsequently, the SRE located in the c-fos promoter has
been shown to mediate increases in chimeric gene
expression during the stretching of cardiac myocytes in
culture (30) and in the pressure-overloaded
heart using
plasmid injection methodology (3).
4007
10000000
8000000
6000000
4000000
2000000
-2090
-424
-202
Actin Promoter
-99
-77
Length
Fig. 4. cx-Actin
chimeric
gene expression,
corrected
for RSVCAT
expression
(see MATERIALS AND METHODS) for each individual
promoter
length in control
and stretched
ALD muscles.
Values are means + SE.
*Significantly
different
from control
(P < 0.05). Inset: chimeric
gene
expression,
corrected
for RSVCAT
expression
for each individual
promoter
length, is given as percentage
difference
between
control
and
stretched
ALD
muscles.
For each promoter
length
mean
control
expression
is set to lOO%, and stretched
value is mean expression
in
stretched
ALD divided by mean expression
in control
ALD.
Actin
Promoter
Length
Fig. 5. Induction
of luciferase
activity
in differentiating
chicken
myoblasts
transfected
with plasmids
containing
indicated
5’ deletion
luciferase
recombinants
and cotransfected
with pCMVgal.
Average
luciferase/P-galactosidase
(GAL) activities
were obtained
from 3 to 5
separate
transfection
experiments
per promoter
deletion.
Transfected
cell cultures
were harvested
and assayed for luciferase
and P-galactosidase activities
72 h after initial plating
as described
in text. Values are
expressed
as means + SE.
ACTIN
PROMOTER
IN
Three SREs are found within -200 bp of the chicken
skeletal a-actin gene transcription
start site and display
nonequivalent
factor-binding
properties, probably due
to different contextual sequences (17). All three SREs
have affinity for the trans-acting DNA binding protein
serum response factor. However, only SREl has binding
affinity for DNA binding protein YY1, which is thought
to repress transcription
of the chicken skeletal cx-actin
promoter by its binding (12, 17). SRE3 does not interact
appreciably with factors showing asymmetric footprints
(17). In cant rast, SREl and SRE2 may behave differently because of the dual factor binding specificity of the
former and the weak SRE affinity of the latter (17).
These observations
could explain why stretch could
activate SREl of the chicken skeletal oc-actin promoter
but not further activate actin promoter activity through
SRE2 and SRE3 in the same promoter.
The SREl in the actin promoter and the SRE in the
c-fos promoter are not equivalent. The skeletal ar-actin
promoter is muscle specific; when the skeletal muscle
SREl is replaced by the c-fos SRE, expression occurs in
both muscle and nonmuscle cells (38). The c-fos SRE is
associated with an ets domain, binding p6ZTcF. A point
mutation at the p62 TCFbinding site of the SRE in the
c-fos promoter abolishes activation of this promoter due
to acute pressure overload of the heart. This demonstrates a role for p6ZTCF in the signaling of stretchinduced increases in expression from the c-fos promoter
(3). SREl of th e ch’ic k en skeletal actin promoter (18)
and several other SREs (35) do not contain a discernible
ets motif, and this has been interpreted
to mean that
p6ZTCF binding may not be common to all SREs (35).
Because of these contextual
differences surrounding
SREs (14,35), it is not certain that results from the c-fos
SRE in heart muscle can be applied to SREl of the
chicken skeletal ar-actin gene.
Our current data suggest that the chicken skeletal
cx-actin gene also contains an important,
but distinct
from the human gene, regulatory element(s) upstream
of the -202-bp promoter (Figs. 4 and 5). Research with
the human skeletal ar-actin promoter has found distal
enhancers - 1,282 to -708 bp from the transcription
start site that increase the expression of the gene lo-fold
(24). It is possible that an additional activator elements
exist from - 202 to - 424 and/or that repressor elements
are present from -424 to -2,090 in the chicken skeletal
ar-actin promoter.
In summary,
our results demonstrate
that direct
plasmid injection techno logy can be combined with
promoter )deletion analysis during stretch-induced hyper-trophy of skeletal muscle in the living animal to determine promoter activity. These data show that chimeric
gene expression from - 2,090-bp chicken ar-actin promoter is increased during hypertrophy
of the ALD
muscle induced by chronic stretch overload. These data
indicate that the gene region responding to stretch is
downstream of -99 and imply, but do not conclusively
prove, that the region between -99 and - 77 contributes
to the stretch-induced
increase in the skeletal ar-actin
promoter in the ALD muscle. Further experime nts will
MUSCLE
c923
HYPERTROPHY
be required
downstream
to determine conclusively which element(s)
of -99 that are responding to stretch.
We thank
Sandra
Higham
and Wei Lou for technical
advice during
the project.
We thank
Marc
Hamilton
for critical
review
of the
manuscript
and Jim Pastore
for expert
assistance
with graphics.
We
also thank
Drs. Samuel Kaplan,
George Winestock,
and M. David Low
for supporting
training
in molecular
biological
techniques.
The research
was supported
by National
Institute
of Arthritis
and
Musculoskeletal
and Skin Diseases
Grant
AR-19393
(F. W. Booth) and
Fellowship
AR-08227
(C. S. Stump).
Address
for reprint
requests:
F. W. Booth, Dept. of Physiology
and
Cell Biology,
Univ.
of Texas-Houston
Health
Science
Center,
6431
Fannin
St., Rm. 4.100 MSB, Houston,
TX 77030.
Received
28 June
1994;
accepted
in final
form
20 October
1994.
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