Induction of Nonmuscle Myosin Heavy Chain II-C by Butyrate in RAW
264.7 Mouse Macrophages*
Denis B.
Buxton
,
Eliahu
Golomb, and
Robert S.
Adelstein
From the Laboratory of Molecular Cardiology, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892
Received for publication, October 3, 2002, and in revised form, February 20, 2003
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ABSTRACT |
RAW 264.7 macrophages express nonmuscle myosin
heavy chain II-A as the only significant nonmuscle myosin heavy chain
isoform, with expression of nonmuscle myosin heavy chain II-B and II-C low or absent. Treatment of the cells with sodium butyrate, an inhibitor of histone deacetylase, led to the dose-dependent
induction of nonmuscle myosin heavy chain II-C. Trichostatin A, another inhibitor of histone deacetylase, also induced nonmuscle myosin heavy
chain II-C. Induction of nonmuscle myosin heavy chain II-C in response
to these histone deacetylase inhibitors was attenuated by mithramycin,
an inhibitor of Sp1 binding to GC-rich DNA sequences. Bacterial
lipopolysaccharide alone had no effect on basal nonmuscle myosin heavy
chain II-C expression, but attenuated butyrate-mediated induction of
nonmuscle myosin heavy chain II-C. The effects of lipopolysaccharide
were mimicked by the nitric oxide donors sodium nitroprusside and
spermine NONOate, suggesting a role for nitric oxide in the
lipopolysaccharide-mediated down-regulation of nonmuscle myosin heavy
chain II-C induction. This was supported by experiments with the
inducible nitric-oxide synthase inhibitor 1400W, which partially
blocked the lipopolysaccharide-mediated attenuation of nonmuscle myosin
heavy chain induction. 8-Bromo-cGMP had no effect on nonmuscle myosin
heavy chain induction, consistent with a cGMP-independent mechanism for
nitric oxide-mediated inhibition of nonmuscle myosin heavy chain II-C induction.
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INTRODUCTION |
Vertebrate nonmuscle myosin II represents a branch of the myosin
superfamily, closely related to skeletal and smooth muscle myosin II
isoforms. This ubiquitously distributed class of myosins has been shown
to be present in all vertebrate cells including smooth muscle, cardiac
muscle, and skeletal muscle cells where, similar to the more highly
expressed isoforms of myosin II, they consist of a hexamer containing
two heavy chains (200 kDa) and two pairs of light chains (20 and 17 kDa). Two isoforms of the nonmuscle myosin heavy chain
(NMHC),1 termed NMHC II-A and
II-B, were identified several years ago (1-3). The genes
encoding these myosin heavy chains are located on different chromosomes
(4) and the tissue distributions of the isoforms differ (4, 5),
although there is considerable overlap, with most cells and tissues
expressing both isoforms to a greater or lesser degree. Recently,
completion of the sequencing of the human genome revealed the presence
of a third nonmuscle myosin, NMHC II-C, which again, has a distinct
chromosomal location compared with the other two isoforms (6).
In hematopoietic cells, NMHC II-A is believed to be the predominant
NMHC isoform; for example, in the rat basophilic leukemia RBL-2H3 cell
line, NMHC II-B was shown to be absent (7). We were, therefore,
interested in whether NMHC II-C is expressed in hematopoietic cells.
Here we demonstrate that expression of NMHC II-C is low or absent in
undifferentiated macrophage RAW 264.7 cells. However, treatment of the
cells with the histone deacetylase (HDAC) inhibitors sodium butyrate or
trichostatin A, which have been shown to lead to differentiation of a
wide range of cell types, results in induction of NMHC II-C. Moreover, induction of NMHC II-C is attenuated by bacterial lipopolysaccharide in
an NO-dependent but cGMP-independent fashion.
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EXPERIMENTAL PROCEDURES |
RAW 264.7 cells were obtained from ATCC (Manassas, VA) and were
cultured in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum. Sodium butyrate, trichostatin A, mithramycin, and
lipopolysaccharide (Escherichia coli serotype 0127:B8) were purchased from Sigma. Sodium nitroprusside, 1400W, 8-bromo-cGMP, and
spermine NONOate were from Calbiochem (La Jolla, CA). Antibodies to
sequences near the carboxyl-terminal end of human NMHC II-C (RQVFRLEEGVASDEEAEE) and near the amino-terminal of mouse NMHC II-C
(VTMSVSGRKVASRPGP) were produced in rabbits and affinity purified
against the corresponding peptides. An antibody to poly(ADP-ribose) polymerase was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
A monoclonal antibody to iNOS was obtained from Transduction Laboratories (Lexington, KY). Nitrate and nitrite were measured using a
kit from Roche Molecular Biochemicals (Indianapolis, IN).
Immunoblotting and Immunoprecipitation--
Cells were washed
twice with phosphate-buffered saline containing 5 mM EDTA
and 1 mM orthovanadate, lysed by addition of lysis buffer,
and the cells were scraped from the plate with a plastic scraper. Lysis
buffer consisted of 25 mM Hepes, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl2, 0.1% Triton X-100, 1 mM sodium orthovanadate, 20 mM
-glycerophosphate, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, 0.15 units/ml aprotinin at 4 °C. Cell
lysates were transferred to a microcentrifuge tube and Triton-insoluble
material pelleted at 14,000 × g for 10 min. After
taking an aliquot for protein determination (8) to permit equal protein
loading of gel lanes, samples were heated in SDS sample buffer
(70 °C, 10 min) and subjected to SDS-PAGE (NuPAGE, Invitrogen).
Samples were transferred to polyvinylidene difluoride membranes
(Immobilon-P, Millipore, Bedford, MA) and immunoblotted using standard
methods. Bound secondary antibody was detected using luminol blotting
reagents (Santa Cruz Biotechnology) and the signal captured on Biomax
MS film (Eastman Kodak, Rochester, NY). Films were scanned using a
laser densitometer (Amersham Biosciences) and bands were
quantified using ImageQuant software. For immunoprecipitation, following centrifugation of lysates and determination of protein content, aliquots of the lysates were rocked for 1 h with the COOH-terminal NMHC II-C antibody. Protein A/G-agarose (Santa Cruz Biotechnology) was then added, and the mixture was rocked overnight at
4 °C. The agarose beads were washed 4 times with ice-cold
phosphate-buffered saline, SDS sample buffer was added, and the samples
were subjected to SDS-PAGE and Western blotting as described above.
RT-PCR--
Total RNA was isolated from cells using Trizol
(Invitrogen). Reverse transcription was carried out on 2 µg of RNA
using Superscript II and random primers (Invitrogen). PCR was then
carried out using Platinum PCR Supermix (Invitrogen), with the cycle
number adjusted to keep product formation in the linear range. For
HPRT, 25 cycles were used and for NMHC II-C, 35 cycles. The primers for
the NMHC II-C PCR were: 5 prime, 5'-gccgaacgaagtttctcag-3'; 3 prime, 5' tcattggggtgtggcagg-3'. The product is a 618-bp fragment corresponding to the last 205 amino acids and the termination codon.
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RESULTS |
We first investigated whether NMHC II-C was expressed in RAW 264.7 macrophages. Immunoblotting of RAW 264.7 cell lysate with an antibody
specific for NMHC II-C demonstrated the myosin isoform to be present at
a very low concentration in the macrophages (Fig. 1A). In contrast, NMHC II-C
was expressed more abundantly in PC12 and COS cells. Treatment of the
RAW 264.7 cells with sodium butyrate for 24 h led to the
dose-dependent induction of NMHC II-C expression (Fig.
1B). A time course of NMHC II-C induction using 10 mM butyrate is shown in Fig. 1C; significant
induction of NMHC II-C was detectable by 10 h of stimulation.
Induction of NMHC II-C was reversible, because removal of butyrate
after 3 days of induction led to a decline in NMHC II-C levels (Fig.
1D). To confirm the identity of the 225-kDa band identified
by the antibody to the COOH terminus of NMHC II-C, immunoblotting was
performed with a second antibody that recognizes a different epitope,
close to the amino terminus of NMHC II-C (see "Experimental
Procedures"). A similar induction of the 225-kDa band was observed
(Fig. 1E), supporting the identity of the induced band as
NMHC II-C.

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Fig. 1.
Induction of NMHC II-C in response to sodium
butyrate in RAW 264.7 macrophages. A, NMHC II-C is very
low in non-stimulated RAW 264.7 cells, but is present in COS-7
fibroblasts and PC12 cells. Immunoblotting in panels
A-D was performed with an antibody to an epitope close
to the carboxyl terminus region of NMHC II-C. Nonspecific bands were
observed at lower molecular weights, particularly in the RAW 264.7 lane; these bands vary in intensity from experiment to experiment, and
band intensity does not correlate with cell treatment. B,
cells were treated with increasing concentrations of sodium butyrate
for 24 h before preparation of cell lysates and immunoblotting for
NMHC II-C. C, time course of induction of NMHC II-C protein
in response to 10 mM sodium butyrate. D,
reversibility of NMHC II-C induction in response to sodium butyrate.
Cells were treated for 3 days in the presence or absence of butyrate, 2 mM. They were then lysed (lanes 1 and
2) or subjected to a change of buffer. Cells that had been
incubated with butyrate were then incubated for a further 3 days with
(lane 4) or without (lane 5) butyrate. Control
cells were incubated without butyrate for a further 3 days (lane
3). N/A, not applicable. E,
immunoblotting of lysates from control and butyrate-treated lysates
with a second antibody recognizing an epitope close to the amino
terminus of NMHC II-C showed a similar induction pattern, supporting
the identity of the induced band as NMHC II-C.
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Analysis of NMHC II-C mRNA expression by RT-PCR demonstrated that
the mRNA level was very low in untreated cells, but was increased
at 6 h and maximal at 10-16 h butyrate treatment (Fig. 2A). RT-PCR of HPRT mRNA
was used as a control, showing that similar mRNA amounts were used.
Induction of NMHC II-C mRNA was not dependent on de novo
protein synthesis, because cycloheximide in the presence of butyrate
did not inhibit induction (Fig. 2B, lanes 5 and
6). In fact, cycloheximide alone induced NMHC II-C mRNA
expression (lanes 3 and 4) and appeared to
enhance butyrate-mediated induction (lanes 5 and
6). Cycloheximide was, however, effective at preventing induction of NMHC II-C protein expression, as demonstrated by immunoblot analysis (Fig. 2C).

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Fig. 2.
Induction of NMHC II-C mRNA in response
to butyrate. A, time course of induction of NMHC II-C
mRNA. Total RNA was subjected to reverse transcription and the
cDNA used for PCR using primers specific for NMHC II-C
(top) or for the housekeeping gene HPRT (bottom).
The left lanes contain DNA markers. B, effect of
cycloheximide on induction of NMHC II-C mRNA. Cells were treated
with cycloheximide, 3.6 or 36 µM, for 30 min and then
incubated with or without sodium butyrate, 10 mM, for
16 h before preparation of RNA and RT-PCR as above. C,
effect of cycloheximide on induction of NMHC II-C protein. Cells were
incubated with or without cycloheximide, 36 µM, for 30 min before incubating in the presence or absence of butyrate, 10 mM, for 24 h. Cells were then lysed and lysates were
subjected to immunoblotting for NMHC II-C. Note the absence of signal
in lane 4 compared with lane 2.
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Sodium butyrate has been shown to inhibit HDAC and the resultant
histone hyperacetylation and chromatin rearrangement have been proposed
as the mechanism by which butyrate activates transcription (9). To
assess the role of HDAC inhibition in NMHC II-C induction, cells were
treated with trichostatin A (TSA), which is also known to inhibit
HDAC. TSA stimulation led to the induction of NMHC II-C and the extent
of induction was similar to that obtained with butyrate (Fig.
3).

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Fig. 3.
Treatment of RAW 264.7 cells with
trichostatin A induces NMHC II-C. A, time course of
NMHC II-C induction in response to TSA, 100 ng/ml (lanes
1-5). Induction in response to 10 mM butyrate for
24 h is shown in lane 6. B, dose response
for induction of NMHC II-C in response to TSA. Cells were incubated for
24 h with the indicated concentrations of TSA before lysis and
immunoblotting for NMHC II-C.
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To determine whether the effects of butyrate on induction of NMHC II-C
were specific for RAW 264.7 cells, the RBL-2H3 rat mast cell line and
HeLa cells were stimulated with butyrate. Non-stimulated RBL-2H3 cells
do not express NMHC II-B (7) and NMHC II-C is also undetectable (Fig.
4A). However, treatment with
butyrate led to a robust induction of expression of NMHC II-C (Fig.
4A). Expression of NMHC II-C is also absent in
non-stimulated HeLa cells (Fig. 4B). Extended treatment for
72 h with high dose butyrate (10 mM) led to a very
modest induction of NMHC II-C, much lower than that found in the
hemopoietic macrophage and mast cell lines. A second slower migrating
band was also recognized by the COOH terminus NMHC II-C antibody (Fig.
4B); however, immunoprecipitation of NMHC II-C with the
carboxyl terminus antibody followed by immunoblotting of the
immunoprecipitate with the mouse NH2 terminus antibody showed only a single band induced by butyrate at 225,000, suggesting that the higher molecular weight band is probably
nonspecific (Fig. 4C).

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Fig. 4.
Butyrate induces NMHC II-C in
RBL-2H3 but only marginally in HeLa cells. RBL-2H3 (A)
and HeLa cells (B) were treated with the indicated
concentrations of butyrate for 72 h before preparation of cell
lysates and immunoblotting for NMHC II-C. An equal loading of lysate
from RAW 264.7 macrophages treated with butyrate (10 mM)
was loaded in the right-hand lane for comparison. For
RBL-2H3 cells, detached cells were collected by centrifugation, washed,
and lysed after pooling with attached cells. An additional band
migrating more slowly than NMHC II-C was observed in the HeLa
experiments. C, immunoprecipitation of lysates prepared from
HeLa cells treated with or without butyrate, 10 mM, for
72 h was performed using the carboxyl terminus NMHC II-C
antibody. Immunoprecipitated proteins were then subjected to
SDS-PAGE followed by immunoblotting with a second NMHC II-C antibody
to an amino terminus epitope. A single band at 225 kDa was observed,
consistent with the slower migrating band in panel B being
nonspecific.
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The transcription factor Sp1 has been implicated in butyrate-mediated
gene regulation (10-13). To address the possible role of Sp1 in
butyrate-mediated induction of NMHC II-C, RAW 264.7 cells were
preincubated with the antibiotic mithramycin, which binds to GC-rich
DNA and inhibits Sp1 binding (14). Mithramycin inhibited induction of
NMHC II-C in a dose-dependent manner (Fig. 5, A and B),
consistent with a role for Sp1 in butyrate-mediated NMHC II-C
induction. A similar mithramycin-mediated inhibition was observed when
TSA was used to induce NMHC II-C (results not shown). Measurement of
NMHC II-C mRNA by RT-PCR demonstrated a similar pattern of
inhibition by mithramycin (Fig. 5C), indicating that the
inhibitor was acting at the transcriptional level.

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Fig. 5.
NMHC II-C induction is attenuated
by the Sp1 inhibitor mithramycin. A, cells were
pretreated with mithramycin, 10 nM to 10 µM,
for 30 min before incubation with sodium butyrate, 10 mM,
for 20 h. B, cells were pretreated with mithramycin, 1 µM, for 30 min before incubation with sodium butyrate, 2 or 10 mM, for 20 h. Cell lysates were then prepared
and subjected to immunoblotting for NMHC II-C. C, cells were
treated with mithramycin for 30 min followed by sodium butyrate for
16 h before preparation of RNA and RT-PCR using primers for NMHC
II-C (top) or HPRT (bottom).
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Butyrate has been shown to modulate the effects of bacterial LPS on
macrophages and endothelial cells, inhibiting iNOS expression (15), but
enhancing expression of interleukin-8 (16) and alkaline phosphatase
(17). It was, therefore, of interest to determine whether there was any
interaction between butyrate and LPS in modulating NMHC II-C isoform
expression. Treatment of naive RAW 264.7 cells with LPS induced
expression of iNOS as expected (Fig. 6A, lower blot,
lane 2), but had no effect on expression of NMHC II-C
(upper blot). However, when LPS was added in combination with butyrate, 2 or 10 mM, induction of NMHC II-C
(upper blot, lanes 4 and 6) was
attenuated. The effects of LPS on NMHC II-C induction are summarized in
Fig. 6B. As reported previously (15), butyrate attenuated
iNOS induction in response to LPS (Fig. 6A, lanes
4 and 6, lower blot).

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Fig. 6.
LPS attenuates NMHC II-C induction.
Cells were treated ± butyrate, 2 or 10 mM, in the
presence or absence of LPS (1 µg/ml) for 24 h. Detached cells
were collected by centrifugation and lysed after pooling with attached
cells. Cell lysates were then subjected to immunoblotting for NMHC II-C
and iNOS (A). B, quantification of the effect of
LPS on butyrate-mediated NMHC II-C induction. Films obtained in three
experiments were scanned and the band intensities were normalized to
induction in response to 10 mM butyrate. Results are
expressed as mean ± S.E. *, p < 0.05 versus butyrate stimulation in the absence of LPS.
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The inhibition of NMHC II-C induction in response to LPS raised the
possibility that the NO produced in response to the induction of iNOS
played a role in regulation of NMHC II-C expression. This was confirmed
by induction of NMHC II-C with butyrate in the presence or absence of
nitric oxide donors. Sodium nitroprusside, 0.25 mM (Fig.
7A), and spermine NONOate, 0.2 mM (Fig. 7B), attenuated NMHC II-C expression in
response to butyrate. The results are summarized in Fig. 7C.
Conversely, the inhibition of butyrate-mediated NMHC II-C induction by
LPS was attenuated by inhibition of iNOS with the specific inhibitor
1400W (Fig. 8). Inhibition of iNOS was
confirmed by measurement of the stable products of NO breakdown, nitrate and nitrite; 1400W inhibited nitrite + nitrate production in
response to LPS by 90% (results not shown).

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Fig. 7.
NMHC II-C induction is inhibited by NO
donors. Cells were treated ± butyrate, 2 or 10 mM, in the presence or absence of sodium nitroprusside,
0.25 mM (A), or spermine NONOate, 0.2 mM (B), for 24 h. Detached cells were
collected by centrifugation and lysed after pooling with attached
cells. Cell lysates were then subjected to immunoblotting for NMHC
II-C. C, quantification of the effect of NO donors on
butyrate-mediated NMHC II-C induction. Results are expressed as
mean ± S.E. for four experiments. *, p < 0.05 versus butyrate stimulation in the absence of NO
donor.
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Fig. 8.
iNOS inhibition blocks the LPS-mediated
inhibition of NMHC II-C induction. Cells were pretreated with the
iNOS inhibitor 1400W, 10 µM, for 30 min before
incubation ± LPS, 1 µg/ml, and butyrate, 2 mM
(lanes 2-5) or 10 mM (lanes 6-9)
for 20 h. Cell lysates were then prepared and immunoblotted for
NMHC II-C (A). B, quantification of the effects
of LPS and iNOS inhibition on butyrate-mediated NMHC II-C induction.
Results are expressed as mean ± S.E. from four experiments. *,
p < 0.05 versus butyrate stimulation in the
absence of LPS. §, p < 0.05 versus
butyrate + LPS.
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NO mediates its effects on vasodilation via activation of guanylate
cyclase and elevation of cGMP, but it can also act in a
cGMP-independent fashion. To address the role of cGMP in the inhibitory
action of NO, induction of NMHC II-C with butyrate was performed in the
presence or absence of a cell-permeable cGMP analog. 8-Bromo-cGMP, 0.1 to 1 mM, had no effect on NMHC II-C induction in response
to butyrate (Fig. 9), arguing against a role for cGMP elevation in the attenuation of NMHC II-C induction by NO
and LPS.

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Fig. 9.
8-Bromo-cGMP does not inhibit
butyrate-mediated NMHC II-C induction. Cells were pretreated with
the indicated concentration of 8-bromo-cGMP for 30 min before
incubation with butyrate, 10 mM, for 20 h. Cell
lysates were then prepared and immunoblotted for NMHC II-C.
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DISCUSSION |
NMHC-II isoforms differ in their tissue distributions, indicating
that expression of the genes is regulated in a cell-specific manner.
Evidence has been obtained previously for transcriptional regulation of
both NMHC II-A and II-B expression. A 100-bp region in intron 1 of
MYH9, the gene encoding human NMHC II-A, located 23 kb downstream from the transcriptional start site, has been shown to
activate transcription in a cell-type and differentiation-specific manner (18). This region contains a binding site for Sp1 or Sp3, a site
for USF1 or USF2, and a novel site (18). Induction of NMHC II-A was
demonstrated during differentiation of HL-60 myeloid cells and U-937
promonocytic cells to a more monocytic phenotype (19). For NMHC II-B,
the homeobox protein Hex has been demonstrated to induce transcription
in a cAMP-response element-dependent manner, although Hex
binds to a specific homeodomain-binding sequence rather than
cAMP-response element (20). NMHC II-B is also down-regulated in cells
transformed by a variety of oncogenes (21).
Here we demonstrate using mouse macrophages that expression of NMHC
II-C can be regulated by a physiologically relevant stimulant, sodium
butyrate. In contrast, this treatment had no effect on the expression
of II-A, which is abundant in these cells. NMHC II-B, which is not
expressed in these cells, similar to RBL-2H3 cells, was only minimally
up-regulated (data not shown). Butyrate is produced in large quantities
by bacterial fermentation of fiber and its intracolonic concentration
is typically in the 5-15 mM range (22). Stimulation with
this short chain fatty acid has been shown to lead to differentiation
in a wide range of cell types and, in many cases, can also cause
apoptosis. A diverse range of gene products have been shown to be
induced by butyrate, including the G-protein G
i2 (13),
cell-cycle related proteins such as p21Waf1 (10, 23) and
p27Kip1 (23),
-globin (24), and alkaline phosphatase
(25). Induction of NMHC II-C was found both in hematopoietic cell
lines, the macrophage line RAW 264.7 and basophilic leukemia line
RBL-2H3, and in non-hematopoietic HeLa fibroblasts. However, HeLa cells
were much less sensitive to butyrate than the hematopoietic lines.
The induction of NMHC II-C mRNA expression does not require
de novo protein synthesis, because cycloheximide, at
concentrations that abolished induction of NMHC II-C protein
expression, tended to increase butyrate-mediated NMHC II-C mRNA
expression. Cycloheximide alone was also able to induce increased
amounts of NMHC II-C mRNA. The induction of specific mRNAs in
response to cycloheximide treatment is often interpreted to indicate
regulation of mRNA expression by a labile regulatory protein (26).
However, other mechanisms by which cycloheximide can act include
stabilization of mRNA (27, 28) and direct transcriptional
activation (29, 30). Cooperation between cycloheximide and TSA in
inducing histone H1 mRNA was proposed to involve a
cycloheximide-mediated rearrangement of chromatin leading to general
transcriptional de-repression (30). However, the current experiments do
not distinguish between these potential mechanisms for NMHC II-C regulation.
Butyrate causes differentiation and cell cycle arrest in a wide range
of cell types (9) and, in many cases, butyrate stimulation also induces
apoptosis. Butyrate inhibits HDAC and the resultant histone
hyperacetylation and chromatin rearrangement have been proposed as the
mechanism by which butyrate activates transcription (9). However, there
is increasing evidence that other mechanisms contribute to the actions
of butyrate. Butyrate stimulation of transcription of several genes,
including p21Waf1 (10), ferritin H (31), galectin-1 (32),
and G
i2 (13), involves Sp1 binding sites. HDAC1 can form
a complex with Sp1 and inhibit transcription through Sp1 binding sites
(33). The transactivating factor E2F1 competes with HDAC1 for binding
to Sp1 and abolishes HDAC1-mediated transcriptional repression (33). Here we show that mithramycin, an antibiotic that binds to GC-rich sequences and blocks binding of Sp1 to DNA (14), inhibited the induction of NMHC II-C in response to butyrate. Inhibition of induction
of NMHC II-C occurred both at the mRNA and protein levels at 1 and
10 µM mithramycin.
The normal condition of the gut has been described as a state of
controlled inflammation (34). Typically, cells of the colon and liver
will be exposed to both butyrate and LPS and the balance of
interactions between these two mediators is likely to play a role in
inflammatory diseases such as irritable bowel syndrome. NMHC II-C
induction is attenuated by LPS in RAW 264.7 cells, an interaction that
could be important in regulating NMHC II-C levels in cells of the colon
and liver. The inhibition of NMHC II-C induction appears to be mediated
by NO, produced in response to the LPS-mediated induction of iNOS,
because the NO donors spermine NONOate and sodium nitroprusside
attenuated NMHC II-C induction in a similar fashion. Conversely,
inhibition of iNOS with the specific inhibitor 1400W attenuated the
LPS-mediated inhibition of NMHC II-C induction. There is, thus, a
mutual antagonism between butyrate and LPS, with butyrate inhibiting
iNOS induction while the product of iNOS, NO, inhibits NMHC II-C induction.
Whereas NO regulates vascular tone via soluble guanylate cyclase (35),
it has been shown to have cGMP-independent effects on gene
transcription. Nitric oxide donors increased tumor necrosis factor-
expression in response to phorbol ester in U937 cells, which lack
soluble guanylate cyclase (11). NO increased tumor necrosis factor-
promoter activity and this increase was associated with decreased Sp1
binding. Insertion of Sp1 sites also conferred NO responsiveness to a
minimal cytomegalovirus promoter (11). NO also inhibited
interleukin-2 expression in response to interleukin-1
in murine
lymphocytes (36). NO was shown to abrogate the DNA binding activities
of the zinc finger transcription factors Sp1 and EGR-1, and native Sp1
derived from NO-treated nuclear extracts and NO-treated lymphocytes
lacked DNA binding activity (36). LPS was found to down-regulate Sp1
binding activity by promoting Sp1 protein dephosphorylation and
degradation (37); whereas the mechanism of LPS action was not
determined, NO would clearly be a candidate effector. The inhibitory
effects of NO on NMHC II-C induction appear to be cGMP-independent,
because the cGMP analog 8-bromo-cGMP had no effect on butyrate-mediated
induction of NMHC II-C. NO regulates NF-
B activity by nitrosylation
of the transcription factor (38), and this has been proposed as a
potential regulatory mechanism for Sp1, perhaps interfering with the
zinc finger structure (39).
It is, thus, tempting to speculate that Sp1 may play a role in both the
induction of NMHC II-C in response to butyrate and in its suppression
by NO. However, confirmation of this hypothesis will require the
isolation of the NMHC II-C promoter, permitting mechanistic studies on
the transcriptional regulation of NMHC II-C.
At higher butyrate concentrations (>2 mM), treatment of
RAW 264.7 cells resulted in the induction of apoptosis, as indicated by
membrane blebbing and poly(ADP-ribose) polymerase
cleavage, and the time course of induction of NMHC II-C
paralleled the onset of apoptosis in response to these relatively
high amounts of butyrate (results not shown). However, induction of
NMHC II-C is not required for apoptosis, because sodium nitroprusside
treatment at concentrations >0.25 mM also caused
apoptosis, but did not induce NMHC II-C protein expression (data not
shown). Conversely, induction of NMHC II-C at lower butyrate
concentrations was not accompanied by apoptosis. Thus, the
function of NMHC II-C isoform induction is currently unknown. Whereas
there is overlap between the tissue distributions and intracellular
localizations of NMHC isoforms, it is clear that the isoforms are not
redundant. Ablation of NMHC II-B expression has been shown to lead to
cardiac (40) and brain abnormalities (41), leading to embryonic or
neonatal lethality, whereas ablation of NMHC II-A leads to lethality at
an early (E6.0) embryonic
stage.2 The II-A and II-B
isoforms also differ in their physical properties; NMHC II-A from
Xenopus had a 2.6-fold greater actin-activated Mg2+-ATPase activity relative to NMHC II-B and moved actin
filaments 3.3-fold faster (42). Further experiments to assess the
physical properties of expressed NMHC II-C and its subcellular
localization may give insight into the role of NMHC II-C and the
functional significance of its induction in response to butyrate.
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ACKNOWLEDGEMENTS |
We thank Mary Anne Conti for reading the
manuscript and acknowledge her help as well as that of Yvette Preston
and Antoine Smith in generating the antibody to human NMHC II-C. We are
grateful to Dr. Robert Wysolmerski for providing anti-mouse NMHC II-C
rabbit serum for affinity purification. We also thank Catherine
Magruder for expert editorial assistance.
 |
FOOTNOTES |
*
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: Heart Research
Program, Two Rockledge Center, 6701 Rockledge Dr., Suite 9044, MSC
7940, Bethesda, MD 20892-7940. Tel.: 301-435-0516; Fax: 301-480-1335; E-mail: db225a@nih.gov.
Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M210145200
2
M. A. Conti and R. S. Adelstein,
unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
NMHC, nonmuscle
myosin heavy chain;
LPS, lipopolysaccharide;
TSA, trichostatin A,
(4,6-dimethyl-7-[p-dimethylaminophenyl]-7-oxohepta-2,4-dienohydroxamic acid);
NO, nitric oxide;
1400W, N-(3-aminomethyl)benzylacetamidine;
spermine NONOate, N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine;
iNOS, inducible nitric-oxide synthase;
HDAC, histone deacetylase;
RT, reverse transcriptase.
 |
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