Distamycin A Inhibits HMGA1-Binding to the P-Selectin
Promoter and Attenuates Lung and Liver Inflammation
during Murine Endotoxemia
Rebecca M. Baron1,2*, Silvia Lopez-Guzman1, Dario F. Riascos1,4, Alvaro A. Macias1, Matthew D. Layne5,
Guiying Cheng6, Cailin Harris1, Su Wol Chung1,7, Raymond Reeves8, Ulrich H. von Andrian6, Mark A.
1Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America,
2Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 3Department of Newborn
Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 4Department of Physiological Sciences, Pontificia
Universidad Javeriana, Bogota, Colombia, 5Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, United States of America, 6CBR
Institute for Biomedical Research and Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America, 7School of Biological Sciences,
University of Ulsan, Ulsan, South Korea, 8Department of Biochemistry and Biophysics, Washington State University, Pullman, Washington, United States of America
Background: The architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich
DNA and forms transcription factor complexes (‘‘enhanceosomes’’) that upregulate expression of select genes within the
inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround
transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such
as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and
HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs.
Objectives: To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue
inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules.
Methodology/Principal Findings: Administration of Dist A significantly decreased lung and liver inflammation during
murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following
an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in
endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist
A disrupted formation of an inducible complex containing NF-kB that binds an AT-rich region of the P-selectin promoter.
Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-kB induction of P-selectin
promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo.
Conclusions/Significance: We describe a novel targeted approach in modulating lung and liver inflammation in vivo during
murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These
studies highlight the ability of MGBs to function as molecular tools for dissecting transcriptional mechanisms in vivo and
suggest alternative treatment approaches for critical illness.
Citation: Baron RM, Lopez-Guzman S, Riascos DF, Macias AA, Layne MD, et al. (2010) Distamycin A Inhibits HMGA1-Binding to the P-Selectin Promoter and
Attenuates Lung and Liver Inflammation during Murine Endotoxemia. PLoS ONE 5(5): e10656. doi:10.1371/journal.pone.0010656
Editor: Mauricio Rojas, Emory University, United States of America
Received September 10, 2009; Accepted April 17, 2010; Published May 14, 2010
Copyright: ? 2010 Baron et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grants AI054465 (to R.M.B.), HL091957 (to R.M.B.), AI061246 (to M.A.P.), GM53249 (to M.A.P.), and HL60788 (to M.A.P.), all
from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Acute lung injury (ALI) represents a devastating clinical
syndrome with increasing incidence that is initiated by an
injurious stimulus, followed by the development of lung inflam-
mation, increased alveolar-capillary barrier permeability, and
influx of protein-rich edema fluid with resultant impairment in gas
exchange due to alveolar flooding. Injury to the lung can be
incurred through direct means (e.g., aspiration pneumonia), or,
more commonly through indirect means (e.g., abdominal sepsis
and resultant bacteremia often from gram negative rods that
elaborate endotoxin). Despite the similar disruption of the
alveolar-capillary membrane as an endpoint of both indirect and
direct lung injury, the underlying mechanisms of injury are likely
quite different, with direct injury initially targeting the lung
alveolar epithelial cell and indirect injury activating the endothe-
lium in the early stages . Irrespective of the mechanism of lung
injury, there exist no targeted treatment strategies for ALI, with
PLoS ONE | www.plosone.org1 May 2010 | Volume 5 | Issue 5 | e10656
current standard of care focusing on supportive approaches [2,3].
Thus novel molecular strategies applied toward improving
outcomes from ALI are desperately needed.
Transmigration of neutrophils into the lung represents a critical
early pathophysiologic step in the development of ALI, as
evidenced by ameliorated lung injury in some animal models in
which neutrophils are eliminated [4,5]. However, application of
anti-inflammatory strategies (e.g., high-dose steroids, cyclooxygen-
ase (COX-2) inhibitors) in ALI treatment has not proven
universally effective [6,7]. Possible reasons for failure of these
approaches are multifactorial [8–10], including the inability to
easily titrate a drug’s effect, i.e., an ‘‘all or none’’ treatment effect.
Therefore, targeted treatment approaches that modulate neutro-
phil migration in a titratable fashion during an inflammatory
stimulus represent important avenues of investigation in ALI. One
potential approach, which we examine herein, is to interfere
predictably with transcription factor-DNA binding to target genes
critical for neutrophil recruitment.
ALI frequently is triggered by bacterial infection, and there exist
an important subset of patients who suffer infections by gram-
negative bacteria. These pathogens elaborate endotoxin (lipopoly-
saccharide, LPS) that triggers a complex inflammatory cascade,
including release of cytokines (e.g., tumor necrosis factor) and
transcriptional up-regulation of numerous genes critical for the
inflammatory response. A number of these cytokine-inducible
genes share common regulatory elements in their promoter
sequences and therefore are up-regulated by common transcrip-
tion factors (TF). TFs such as NF-kB, IRF-1, and Stat-1 have been
implicated as important regulators of a number of inducible genes
in the inflammatory pathway, including P-selectin, E-selectin,
vascular cell adhesion molecule (VCAM)-1, and nitric oxide
synthase (NOS)-2 [11–16]. Specifically, P-selectin is upregulated
by both LPS and TNF-a via similar transcriptional mechanisms
[12,17–19]. Beyond the simplified paradigm of TF-DNA binding
leading to gene activation, elegant molecular studies have
demonstrated that larger complexes of multiple transcription
factors interacting with each other, as well as with the conserved
DNA promoter motifs, are important for gene regulation and have
been termed ‘‘enhanceosomes’’ .
Enhanceosome formation is facilitated by a group of proteins
known as architectural transcription factors that are critical for gene
regulation due to their ability to modify DNA conformation and to
recruit DNA-binding of other TFs [21,22]. High mobility group A1
(HMGA1, formerly known as HMG-I/Y) is an architectural
transcription factor that binds to AT-rich DNA in the minor
groove via three ‘‘AT-hook’’ DNA-binding motifs. HMGA1
binding sites are often adjacent to or overlap with consensus
binding sites for conventional TFs. The role of HMGA1 in
enhanceosome formation has been studied most extensively in
regulating expression of the virus-inducible interferon (IFN)-b gene
[20,23,24]. Similarly, HMGA1 plays a critical role in facilitating
binding of nuclear factor (NF)-kB to the human E-selectin promoter
[14,15], and as demonstrated by our laboratory, to the NOS2
promoter [25,26], to enable transcriptional up-regulation of these
genes following inflammatory cytokine induction. Thus AT-rich
regions surrounding TF consensus binding sites within promoters of
a number of genes within the inflammatory cascade play critical
roles inthe inflammatoryresponse. The ability to interferewithAT-
rich region DNA binding in a predictable fashion therefore has
significant potential to regulate a subset of genes and modulate the
Minor groove binding drugs (MGBs), including the antibiotic
Distamycin A (Dist A), constitute a class of drugs that bind AT-rich
sequences within the minor groove of DNA in a sequence- and
conformation-specific fashion, thereby interfering with TF-DNA
binding to AT-rich sequences [27,28]. HMGA1 is one of the few
transcription factors known to bind exclusively to AT-rich DNA in
the minor groove [29,30]. Therefore, our group and other
investigators have used MGBs to study the effect of interfering
with HMGA1-DNA binding in vitro [26,31,32]. In order to
determine whether MGBs might modulate gene expression in a
similar fashion in vivo, we examined the effect of MGBs on
mortality and hypotension during murine endotoxemia . Dist
A conferred a significant survival benefit following intraperitoneal
LPS and attenuated the hypotensive response during murine
endotoxemia. This beneficial effect in vivo correlated with
attenuation of NOS2 induction in tissues and in murine
macrophages. Furthermore MGBs interfered specifically with
TF-DNA binding in a selective fashion to a distinct AT-rich region
of the NOS2 enhancer. Thus, the ability to regulate transcription
of targeted genes during an inflammatory state represents a novel
and powerful tool toward development of potential therapeutics.
Given the presence of similar regulatory regions in the
promoters of the inducible genes E-selectin and P-selectin and
their roles in neutrophil recruitment to the tissues, we now
hypothesize that MGBs might likewise affect transcriptional
regulation of these genes. Thereby, attenuated neutrophil
recruitment to the tissue might account for the beneficial effect
of MGBs in vivo, and MGBs may therefore represent a new class of
anti-inflammatory molecules. To test this hypothesis, we examined
the effect of MGBs on neutrophil recruitment during murine
endotoxemia. We analyzed the effect of MGBs on neutrophil-
endothelial interactions in vivo, followed by testing of the effects of
MGBs on expression and promoter trans-activation of candidate
genes (P-selectin, E-selectin) involved in the distinct steps of the
inflammatory cascade. Furthermore, the effects of MGBs on
DNA-protein interactions were characterized and revealed that
HMGA1-DNA binding is critical for full induction of the P-
selectin promoter, and, moreover, that inhibition of HMGA1-
DNA binding in vivo at a novel AT-rich DNA site within the P-
selectin promoter correlates with attenuated inflammation during
Male C57BL/6 wild-type (WT) mice (Charles River Laborato-
ries, 6–8 weeks of age) were injected with lipopolysaccharide (LPS)
40 mg/kg (Escherichia coli serotype O26:B6 endotoxin, Sigma) or
vehicle (saline) intraperitoneally (i.p.). Mice also received Dis-
tamycin A (25 mg/kg) i.p. 30 minutes prior to LPS administration
(Dist A, Sigma) or Vehicle (dimethylsulfoxide, DMSO mixed with
PBS, Sigma) as described previously . RNA was extracted
from lung tissue 2 hours following LPS treatment . In separate
experiments, lung and liver tissue was processed for immunohis-
tochemistry between 4 and 24 hours after LPS treatment and
stained for Gr-1 (neutrophils) (Pharmingen), or P-selectin (Santa
Cruz Biotechnology) . All research involving animals was
conducted according to the recommendations for the ‘‘Guide for
the Care and Use of Laboratory Animals’’, and all animal studies
were approved by the Harvard Medical Area Institutional Animal
Care and Use Committee (IACUC). Animals were housed in
pathogen-free barrier facilities and regularly monitored by the
Cell culture and reagents
Bovine aortic endothelial cells (BAEC) and primary murine lung
endothelial cells (MLEC) (generous gift of Dr. Augustine Choi)
HMGA1 and Inflammation
PLoS ONE | www.plosone.org 2May 2010 | Volume 5 | Issue 5 | e10656
were isolated and cultured as described previously [35,36]. Murine
bEnd.3 endothelial cells (American Type Culture Collection) were
cultured as recommended. Human and murine recombinant
tumor necrosis factor (TNF)-a were obtained from PeproTech Inc.
(Rocky Hill, NJ).
The mouse [21379/213] P-selectin luciferase reporter plasmid
(mp1379LUC, cloned into p0LUC) was a generous gift of Rodger
P. McEver . The human [2578/+35] E-selectin pCAT3
reporter plasmid was a generous gift of Tucker Collins . The
E-selectin promoter sequence was subcloned into the Acc65I/
XhoI sites of pGL2-Basic (Promega) , resulting in generation
of a plasmid construct termed (Esel-luc). The human dominant-
negative HMGA1 cDNA construct (mutant HMGI(mII,mIII))
lacks the ability to bind AT-rich DNA sequences in vitro but retains
capacity for specific protein-protein interactions with other
transcription factors . This mutant construct was subcloned
into the HindIII/KpnI sites of the pCMVFlag expression vector
(Sigma-Aldrich Co., St. Louis, MO) with optimization of the
Kozak consensus sequence  (CTTATG to GCCATG),
resulting in generationof
(DNHMGA1-pCMVFlag) . The p50, p65, and HMGA1
expression vectors were generated through cloning full-length
cDNA sequences  into pcDNA3 (Invitrogen). Constructs were
confirmed by sequencing, and where appropriate, expression was
tested using the TNT T7 Quick Coupled Transcription/
Translation System (Promega Corporation, Madison, WI).
a plasmid constructtermed
Transient transfections of BAEC cells and reporter assays
P-selectin and E-selectin plasmids (1.0 mg) were transiently
transfected into BAEC cells using FuGENE 6 transfection reagent
(Roche Applied Science), as described previously . Twelve
hours following transfection of the reporter construct and a b-
galactosidase expression vector (to normalize for luciferase
activity), cells were conditioned in standard media containing
2% fetal bovine serum (FBS), then pre-treated with Dist A (25 mM)
or Vehicle (ethanol, less than 1% final volume), followed by
addition of LPS (1 mg/ml) or human TNF-a (10 ng/ml) 30
minutes later. Following treatment, cells were harvested 4 hours
after LPS treatment and 12 hours after TNF-a treatment and
assayed for luciferase activity (Promega Luciferase Assay System)
and b-galactosidase . In separate experiments, transient
transfections were undertaken with the P-selectin promoter
(0.5 mg), increasing concentrations of the DNHMGA1-pCMVFlag
vector (0.5–1.0 mg, or an empty vector as a control), p50/p65 and
HMGA1 expression vectors (0.25 mg each), and a b-galactosidase
expression vector (to normalize for luciferase activity).
RNA isolation and Northern blot analysis
RNeasy Mini RNA isolation kit (Qiagen) was used to extract
total RNA from mouse tissues according to the manufacturer’s
instructions. Northern blot analysis using a radiolabeled murine P-
selectin probe (generous gift of Rodger P. McEver ), E-selectin
probe (generous gift of Mukesh Jain ), or HMGA1 probe
[26,42] was performed as previously described . A radiola-
beled rRNA 18S probe  was used to confirm equal loading.
Quantitation of message for each gene relative to 18S was
undertaken using ImageQuant software (GE Healthcare).
Electrophoretic Mobility Shift Assay (EMSA)
EMSAs were performed as described previously  with
double-stranded oligonucleotide probes encoding an AT-rich
sequence within a region previously demonstrated to be critical
for induction of the murine P-selectin promoter [(2542 to 2521):
59-AGAAATTCTCCCTGGATTTTCC-39] . Nuclear ex-
tracts were harvested from BAEC cells or primary murine lung
endothelial cells with or without 1 hour of exposure to human
TNF-a or murine TNF-a (10 ng/ml), and nuclear protein was
quantified by the Bradford dye-binding method (Bio-Rad).
HMGA1 peptide (43 amino acids) was synthesized by Tufts
Physiology Dept Core Facility (Boston, MA) and encompassed the
AT-hook DNA binding domains (DBD)-2 and DBD-3 . The
radiolabeled probes were incubated with 10–20 mM Dist A (or
ethanol as a vehicle control) for two hours prior to electrophoresis.
In separate experiments to test for presence of specific proteins
within the TNF-a-inducible complex, the nuclear protein mixture
was incubated for 30 min at room temperature with antibodies
against NF-kB family members (p50 and p65, Santa Cruz), Ets-1
(unrelated antibody, Santa Cruz), or an isotype control IgG (Santa
Chromatin Immunoprecipitation (ChIP)
ChIP analysis was performed as described previously  using
the Chromatin Immunoprecipitation Assay Kit (Millipore) on
bEnd.3 cells. The protocol was carried out according to the
manufacturer’s instructions, using approximately 26106cells
harvested 3 hours after treatment with 10 ng/ml murine TNF-a
and/or Dist A (50 mM, or the appropriate Vehicle control).
Following formaldehyde crosslinking, cell lysates were sonicated 25
times for 15 sec each time to shear the genomic DNA to 200–
1000 bp lengths. Immunoprecipitation was subsequently carried
out with either an HMGA1 affinity-purified antibody  or an
equivalent amount of rabbit IgG control. Following reversal of
formaldehyde crosslinking, genomic DNA was purified using
QIAquick PCR Purification Kit (Qiagen). For the positive control
sample (‘‘input’’), a 1% volume of sample was removed before the
immunoprecipitation step, followed by subsequent reversal of
crosslinks and DNA purification as described. Precipitated (and
‘‘input’’ control) DNA was subjected to 35 cycles of PCR using
primers to amplify a 246-basepair region of the murine P-selectin
promoter (encompassing the AT-rich region at basepairs 2542 to
2521 described above) [basepairs 2699 to 2453; Forward
verse primer: 59-GGATGCCAGAGAATGGTTAAA-39] or ap-
proximately 200-basepair regions of the murine P-selectin coding
region and upstream promoter region as negative controls in
CCGCTTTCGTTTAAAACAGG-39; Upstream promoter re-
gion-Forward primer: 59-TTGTACCAACCTATGTAATTTCA-
TAGT-39]. Quantitation of precipitated DNA relative to input
DNA was undertaken using Quantity One 1-D Analysis Software
Intravital Microscopy (IVM) and Image Analysis
IVM and analysis was performed as described previously .
Briefly, mice were treated with i.p. Dist A (25 mg/kg, or Vehicle
control (DMSO/PBS)) followed 30 min later by intrascrotal
murine TNF-a injection (500 ng). Mice were then anesthetized
with i.p. ketamine/xylazine, and the right cremaster muscle was
prepared as previously described  and overlaid with sterile,
bicarbonate-buffered Ringer’s injection solution (pH 7.4). Fluor-
escently labeled endogenous circulating leukocytes (achieved
through injection of 2 mg/ml rhodamine 6G via internal jugular
vein catheter insertion) were visualized by an X40 water-
HMGA1 and Inflammation
PLoS ONE | www.plosone.org3May 2010 | Volume 5 | Issue 5 | e10656
immersion objective (Zeiss Acroplan NA 0.75 ‘; Oberkochen,
Germany) by video-triggered stroboscopic epi-illumination on an
intravital microscope (IV-500; Mikron Instruments, San Marcos,
CA). At least three venule trees per mouse were chosen, and 1-min
recordings were collected of sub-segments of postcapillary and
small collecting venules at 5-min intervals to assess baseline rolling.
Measurements were taken hourly for four hours following TNF-a
injection. The rolling fraction for each individual venule was
calculated as percent leukocytes interacting with the vessel wall
amongst total number of detected fluorescent cells passing the
vessel during the observed period. Sticking efficiency is defined as
the percentage of cells that engage in firm arrest ($30 sec) among
all leukocytes passing a microvessel during the analysis interval.
Vessel cross-sectional diameters, velocities of individual rolling and
non-interacting leukocytes, wall shear rate, and wall shear stress
were determined as previously described .
Results for each treatment group are summarized as mean
values 6 standard error (SE). Comparison of results among
multiple groups at different time points was performed by two-way
analysis of variance (StatMost Software, Salt Lake City, UT).
Comparisons between means of two groups were performed using
an unpaired t-test. Statistical significance was defined as a p
Distamycin A Attenuates Lung and Liver Endotoxin-
Induced Lung Inflammation
To examine the effect of Dist A on inflammation during
systemic endotoxemia, C57BL/6 male adult mice were treated
intraperitoneally (i.p.) with vehicle, LPS/Vehicle or LPS/Dist A
(n=9 per treatment group). Lung (Fig. 1A) and liver (Fig. 1B)
tissue were harvested following treatment, processed for immuno-
histochemistry, and subjected to Gr-1 (neutrophil) staining. The
number of positively stained cells in the Vehicle, LPS/Vehicle and
LPS/Dist A groups was quantified using determination of brown
pixilated area by NIH Image Software. As reported by our group
and others, systemic endotoxin (LPS/Vehicle) results in recruit-
ment of inflammatory cells to the lung interstitium [47,48] and
liver parenchyma [49–51] (p,0.05 compared with vehicle-treated
mice in Fig. 1A–B). Interestingly, treatment with Dist A decreased
endotoxin-induced lung inflammation at 4 hours following
treatment, and this reduction remained at 24 hours following
treatment (0.6160.06 vs 0.3860.05%Area per 2006 field for
LPS/Vehicle vs LPS/Dist A, respectively; p,0.05). Treatment
with Dist A significantly decreased liver inflammation at four
hours following treatment (0.2560.04 vs 0.1760.04%Area per
2006field for LPS/Vehicle vs LPS/Dist A, respectively, p,0.05),
and this trendremained
0.0660.009%Area per 2006 field for LPS/Vehicle vs LPS/Dist
A, respectively), though with an overall reduction in Gr-1 staining
in all groups at 24 hours compared with the 4-hour timepoint.
Distamycin A Attenuates Inflammatory Cytokine-Induced
We hypothesized that the effect of Dist A in attenuating
endotoxin-induced lung and liver inflammatory cell recruitment
would correlate with reduced interactions of circulating neutro-
phils with the endothelial surface. To test this hypothesis, we
subjected mice to intravital microscopy of the cremasteric muscle
at 3–4 hours following treatment with systemic TNF-a/Dist A or
TNF-a/Vehicle (Representative Still Photos, n=3 mice per
treatment group, Figure 2A). TNF-a was selected for these
experiments as representing a key pro-inflammatory cytokine in
the LPS pathway. Preliminary experiments and intravital
microscopy analysis demonstrated that total circulating cell counts
were not altered by administration of Dist A alone (data not
shown). However, the number of adherent cells to the endothelial
surface was visibly reduced with the addition of DistA during a
systemic inflammatory response, and formal analysis of the distinct
phases of neutrophil-endothelial interaction  revealed a
significant reduction in the rolling fraction (39.462.8% vs
23.262.5%, p=0.0001) and sticking efficiency (21.363.5% vs
8.561.5%, p=0.0004) in the TNF-a/Dist A-treated mice when
compared with the TNF-a/Vehicle-treated mice (Fig. 2B).
Distamycin A Selectively Decreases Induction of P-
selectin Expression and Promoter Activity
Given the role of the minor groove binder Dist A in decreasing
rolling fraction and sticking efficiency of leukocytes in TNF-a-
treated mice, we hypothesized that inducible expression and
promoter activity of cytokine-induced genes critical for this effect
would be reduced in the presence of Dist A. In considering the
molecules critical for early neutrophil-endothelial interactions
, we selected two adhesion molecules demonstrated previously
to be inducible by cytokines in endothelial cells: P-selectin  and
E-selectin . We hypothesized that reduction of P-selectin
message would be mediated at the transcriptional level and,
therefore, that induction of P-selectin promoter activity would be
selectively attenuated by Dist A. To test the effect of Dist A on
cytokine induction of P- and E-selectin promoter activities, we
performed transient transfections in bovine aortic endothelial cells
(BAEC) using promoter-reporter constructs for each of these
genes. Transfected cells were then treated with Vehicle, TNF-a/
Vehicle, or TNF-a/Dist A; or with Vehicle, LPS/Vehicle, or
LPS/Dist A and assessed for luciferase activity (with normalization
for b-galactosidase activity) twelve hours or four hours after
transfection, respectively (Figure 3A). As anticipated [12,53], both
genes exhibited inducible promoter activity following treatment
with TNF-a and LPS. Interestingly, while inducible promoter
activity of E-selectin was not significantly altered with the addition
of Dist A to TNF-a or LPS (p=NS), the induction of P-selectin
promoter activity was markedly attenuated when transfected cells
were treated with TNF-a/Dist A or LPS/Dist A as compared with
TNF-a/Vehicle (78% reduction, p=0.006) or LPS/Vehicle (52%
reduction, p=0.004). Given the critical importance of P-selectin in
the rolling fraction and sticking efficiency phases of the adhesion
cascade [52,54], these findings raise the important possibility that
Dist A mediates reduced neutrophil-endothelial interaction
through transcriptional down-regulation of P-selectin promoter
activity. Moreover, in contrast to P-selectin, and similar to the
findings of other investigators , cytokine-induced E-selectin
promoter activity is not significantly altered in the presence of Dist
A. These results lend further support to the importance of P-
selectin in mediating the observed effects of Dist A on attenuated
neutrophil recruitment in our model.
To assess the effect of Dist A on inducible expression of these
genes, lung tissues were harvested from mice at two hours
following treatment with Vehicle, LPS/Vehicle, or LPS/Dist A.
Tissues were then subjected to RNA extraction and Northern-
blotting using radiolabeled P-selectin or E-selectin probes (and an
18S probe as a loading control) (Fig. 3B). While both P-selectin
and E-selectin expression increased following LPS treatment
(21.561.5 fold for P-selectin and 2.061.5-fold E-selectin), only
P-selectin expression was substantially attenuated following Dist A
HMGA1 and Inflammation
PLoS ONE | www.plosone.org4May 2010 | Volume 5 | Issue 5 | e10656
Figure 1. Distamycin A attenuates endotoxin-induced lung and liver inflammation. A. Lungs were harvested from C57BL/6 male mice
24 hours following treatment with Vehicle (Veh), LPS/Vehicle (LPS+Veh) or LPS/Dist A (LPS+Dist A) i.p. (n=9 mice per group). Lungs were fixed,
processed, sectioned, and stained for Gr-1 (a marker of neutrophils, positive staining indicated by brown cells). Representative sections for each
condition are shown (2006 magnification). Using NIH Image Software, 5 fields (at 2006 magnification) per lung section from mice from each
treatment group were quantified, with brown pixels counted as positive staining. Results were expressed as % positively stained area per 2006field.
(*p,0.05 compared with Veh; **p,0.05 compared with LPS+Veh). B. Liver tissue was harvested from C57BL/6 mice 4 h after treatment with Vehicle
(Veh), LPS/Vehicle (LPS+Veh) or LPS/Dist A (LPS+DistA) i.p., (n=at least 4 mice per group) then processed, stained for Gr-1 (neutrophil marker), and
analyzed as described above for Fig. 1A. (*p,0.05 compared with Veh; **p,0.05 compared with LPS+Veh).
HMGA1 and Inflammation
PLoS ONE | www.plosone.org5 May 2010 | Volume 5 | Issue 5 | e10656
treatment (26.063.2% reduction for P-selectin vs 4.060.005%
reduction for E-selectin).
Distamycin A Attenuates P-selectin Tissue Expression in
Lung and Liver During Endotoxemia
Given the presence of P-selectin expression within vascular
endothelial cells as well as within platelets, we examined lung and
liver tissue sections for the effect of Dist A on inducible P-selectin
expression within the lung and liver parenchyma that has been
described by others during endotoxemia [49–51]. Lung and liver
tissue was harvested from mice following treatment with Vehicle,
LPS/Vehicle, or LPS/DistA. Lung (Fig. 4A) and liver (Fig. 4B)
tissues were then subjected to immunohistochemistry using a P-
selectin antibody. Analysis of lung sections revealed a significant
LPS-induced increase in P-selectin staining within the lung
vasculature that was reduced in the presence of Dist A at 4 hours
after treatment, and this reduction remained at 24 hours after
treatment (p,0.05 compared with LPS/Vehicle slides). Similarly,
significant reduction in P-selectin staining in the liver was seen
with LPS/DistA compared with LPS alone at 4 hours after
treatment (0.2960.02 vs 0.1160.02%Area per 2006 field for
LPS/Vehicle vs LPS/Dist A, respectively, p,0.05). By 24 hours
0.0160.005%Area per 2006 field for LPS/Vehicle vs LPS/Dist
A, respectively, p=NS). Notably, there was a significant overall
reduction of P-selectin staining in all groups at 24 hours after
treatment, compared with the four-hour timepoint.
Distamycin A disrupts binding of an inducible protein-
DNA complex containing NF-kB to an AT-rich region of
the P-selectin promoter
Given the effects of the minor groove binder Dist A in inhibiting
inducible P-selectin expression in tissues and promoter-activity in
endothelial cells, we next tested the hypothesis that Dist A
decreases protein-DNA binding to the P-selectin promoter. We
first examined HMGA1 expression in lung tissue of mice following
vehicle, LPS/vehicle, or LPS/DistA to determine whether Dist A
directly affected HMGA1 message levels (Fig. 5A). We hybridized
the same blot as in Fig. 3B for HMGA1, and we found no
significant effect of LPS or DistA on HMGA1 expression
(1.2560.075 fold change for LPS/veh vs vehicle and 1.3160.25
fold change for LPS/DistA vs vehicle, respectively; p=NS). Next,
to test DNA-protein binding activity, we examined an AT-rich
DNA region within the P-selectin promoter that has previously
been demonstrated to be critical for induction of promoter activity
(basepairs 2542 to 2521). Pan et. al. demonstrated that members
of the NF-kB family (p50/p65 subunits) are part of an inducible
binding complex that forms within this promoter region .
Moreover, these authors speculated that HMGA1 might also bind
this AT-rich region and facilitate NF-kB-binding through a
mechanism similar to that described for upregulation of a number
of other genes in the inflammatory cascade, including NOS2 and
E-selectin [13,14,20,24,25]. We therefore set out to determine
whether Dist A would disrupt a previously described inducible
binding complex that forms at an AT-rich region of the P-selectin
promoter and, moreover, whether HMGA1 binds with NF-kB
family members to this AT-rich region.
Nuclear extracts harvested from BAEC cells or primary murine
lung endothelial cells (MLEC) two hours following treatment with
vehicle or TNF-a (human TNF-a for BAECs and murine TNF-a
for MLECs) were electrophoresed with radiolabeled probes
spanning the AT-rich region (basepairs 2542 to 2521) from the
P-selectin promoter (Figure 5). As described previously , an
Figure 2. Distamycin A attenuates inflammatory cytokine-
induced neutrophil-endothelial interactions. A. Wild type mice
were subjected to intravital microscopy analysis at 3–4 hours following
treatment with TNF-a/DistA or TNF-a/Vehicle (Veh) (n=3 mice per
treatment group with multiple vessels and vascular segments analyzed
for each animal). Representative still photos of vascular segments are
shown for the 4-hour time point. White round spots lining the vascular
wall represent adherent leukocytes. B. Formal video analysis of the
phases of neutrophil-endothelial interaction was performed, and values
for all vascular segments within a particular treatment group were
averaged for each time point. A significant reduction in rolling fraction
and sticking efficiency was detected in the TNF-a/Dist A mice at 3–
4 hours (p=0.0001 and 0.0004, respectively), compared with the TNF-a/
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inducible ‘‘doublet’’ complex was observed in BAEC and MLEC
cells following treatment with TNF-a (Fig. 5B lanes 3 and 9–10
and Fig. 5C, lane 3). Next, Dist A (or Vehicle control, V) was
incubated with the radiolabeled probe prior to gel electrophoresis.
Dist A decreased binding within the TNF-a-inducible complex,
particularly the upper band of the ‘‘doublet’’ (Fig. 5B, lanes 4-5,
11–12), and use of identical and non-identical cold competitors
(IC, NIC) confirmed the specificity of this inducible complex, with
elimination of the binding complex with the IC (Fig. 5B, lane 6)
and retention of the inducible complex with the NIC (Fig. 5B, lane
Incubation of the nuclear extracts from TNF-a-treated cells
with NF-kB family member antibodies (p50 and p65) revealed
presence of these proteins in the complex as indicated by
supershifted and/or disrupted bands (Fig. 5C, lanes 4–5), while
no supershift or disruption of the binding complex was observed
with an unrelated antibody (Ets-1, Fig. 5C, lane 6) or an isotype
control antibody (IgG, Fig. 5C, lane 7).
HMGA1 is critical for induction of P-selectin promoter
Given that Dist A selectively decreases P-selectin expression and
promoter activity (Fig. 3–4) and, moreover, that Dist A disrupts
formation of an inducible binding complex at an AT-rich region of
the P-selectin promoter containing HMGA1 and NF-kB (Fig. 5),
we hypothesized that HMGA1 binds to the P-selectin promoter in
this region and plays a critical role in P-selectin induction.
Conversely, inhibition of HMGA1 binding would therefore be
expected to attenuate induction of P-selectin promoter activity. To
test this hypothesis, we examined whether HMGA1 would
facilitate NF-kB induction of the P-selectin promoter (Fig. 6A)
and, conversely, whether a dominant-negative form of HMGA1
Figure 3. Distamycin A selectively decreases induction of P-selectin promoter activity and expression. A. BAEC cells were transiently
transfected with promoter-reporter constructs for P-selectin and E-selectin. Transfected cells were treated with Vehicle, TNF-a/Vehicle, or TNF-a/Dist
A, then harvested twelve hours after treatment and assessed for luciferase activity (with normalization for b-galactosidase levels). Similar experiments
were performed in which transfected cells were treated with Vehicle, LPS/Vehicle, or LPS/Dist A and harvested four hours after treatment. Fold
change was assessed relative to normalized values of ‘‘1’’ for the Vehicle-treated condition for each construct. These experiments were repeated 3
separate times with duplicate wells for each condition. (*p,0.05 compared with Vehicle for each construct; **p,0.05 compared with TNF-a/Vehicle
or LPS/Vehicle for P-selectin). B. Lung tissue was harvested from wild type mice two hours following treatment with Vehicle (Veh), LPS/Vehicle, or
LPS/Dist A (Dist), then subjected to RNA extraction and Northern blotting using a radiolabeled probe for P-selectin or E-selectin (and an 18S probe as
loading control). This experiment was repeated two separate times.
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PLoS ONE | www.plosone.org7 May 2010 | Volume 5 | Issue 5 | e10656
that does not bind DNA (DN-HMGA1)  would decrease
TNF-a-induced P-selectin promoter activity (Fig. 6B).
BAEC cells were first transiently transfected with a P-selectin
promoter-reporter construct with addition of expression vectors
for NF-kB family members (p50/p65) and HMGA1 (Fig. 6A). No
significant change in basal P-selectin promoter activity was seen
with addition of HMGA1 alone, as has been described previously
with other genes . Addition of p50/p65 resulted in significant
upregulation of the P-selectin promoter (p,0.05 vs. P-selectin
promoter alone). Interestingly, the addition of HMGA1 along with
p50/p65 resulted in synergistic upregulation of the P-selectin
promoter, beyond that of p50/p65 alone (p,0.05 vs. P-selectin
promoter alone). Thus, HMGA1 facilitates upregulation of P-
selectin promoter activity by p50/p65.
To investigate the role of HMGA1 in upregulating P-selectin
promoter activity, BAEC cells were transiently transfected with a
P-selectin promoter-reporter construct and increasing concentra-
tions of DN-HMGA1 (or empty vector control) (Fig. 6B).
Transfected cells were treated with TNF-a, then harvested to
assess for P-selectin promoter activity. We observed a significant
reduction in TNF-a-induced P-selectin promoter activity in a
dose-dependent fashion with increasing concentrations of DN-
HMGA1 (p,0.05 for 0.5 mg and 1.0 mg DN-HMGA1 when
compared with empty vector control). Of note, the DN-HMGA1
construct had no significant effect on the promoter activity of
another unrelated gene (heme oxygenase-1, data not shown).
Thus, HMGA1 is critical for TNF-a-induced P-selectin promoter
Distamycin A blocks in vitro and in vivo HMGA1 binding
to a distinct AT-rich region of the P-selectin promoter
We next set out to further test the hypothesis that Dist A
attenuates induction of P-selectin promoter activity through
inhibiting binding of HMGA1 to an AT-rich DNA region. To
do this, we carried out electrophoretic mobility shift assays
(EMSAs) using a synthesized protein (to assess in vitro binding,
Fig. 6C) and chromatin immunoprecipitation experiments using
murine endothelial cells (to assess in vivo binding, Fig. 6D). First,
EMSAs were performed using HMGA1 peptide (containing the
AT-hook DNA binding domains, see Methods) and a radiolabeled
probe spanning the AT-rich region of the P-selectin promoter
(basepairs 2542 to 2521, as above). Significant binding of the
HMGA1 peptide to this site was observed (Fig. 6C, lane 2), while
addition of Dist A significantly reduced in vitro binding of the
HMGA1 peptide (Fig. 6C, lane 3).
Next, to examine in vivo binding of HMGA1 to the AT-rich
region of the P-selectin promoter, chromatin immunoprecipitation
(ChIP) was performed using murine endothelial cells (bEnd.3) and
an affinity-purified HMGA1 antibody . We and others have
described that HMGA1 can function as an architectural
transcription factor that binds constitutively to AT-rich DNA.
With cytokine treatment, HMGA1 then facilitates binding of other
transcription factors to form an inducible transcription factor
complex, or enhanceosome . Therefore, we hypothesized that
HMGA1 would bind the P-selectin promoter pre- and post-TNF-a
treatment, while addition of Dist A would inhibit HMGA1 binding
in vivo. To test this hypothesis, ChIP was performed on cells treated
with vehicle, TNF-a/Vehicle, and TNF-a/Dist A (Fig. 6D).
Immunoprecipitated DNA (as well as ‘‘input’’ control DNA) was
amplified by PCR with primers spanning the AT-region of the P-
selectin promoter described above, with presence of a band
indicating in vivo binding of the designated protein to the amplified
DNA segment. No significant in vivo DNA-protein binding was
detected with use of the IgG control antibody (Fig. 6D, lanes 1–2
in lower panel) or in immunoprecipitated (IP) DNA with primers
amplifying an upstream promoter region or coding region of the
P-selectin gene in vehicle-treated cells (upper panel, Fig. 6D). In
contrast, HMGA1 in vivo binding was detected in cells treated with
Vehicle (‘‘Promoter’’ in upper panel and lane 3 in lower panel;
p,0.05 compared with IgG control Ab), TNF-a (lane 4, lower
panel), and TNF-a/Vehicle (lane 6, lower panel). Interestingly,
Figure 4. Distamycin A decreases lung and liver P-selectin
tissue staining. A. Representative sections (2006 magnification) of
lung tissue harvested from C57BL/6 mice 24 h after treatment with
Vehicle (Veh), LPS+Veh, or LPS+Dist A (Dist). Tissue was fixed, processed,
and stained with a P-selectin antibody. Number of positively stained
vessels was counted in sections from at least 5 mice in each treatment
group. (Arrows demonstrate examples of positively stained vessels;
*p,0.05 compared with Veh; **p,0.05 compared with LPS/Veh). B.
Representative sections (2006 magnification) of liver tissue harvested
from mice 4 h after treatment with Vehicle (Veh), LPS+Vehicle (Veh) or
LPS+Dist A (Dist). Tissue was fixed, processed, and stained with a P-
selectin antibody. Using NIH Image Software, 5 fields (at 2006
magnification) per liver section from mice from each treatment group
were quantified, with brown pixels counted as positive staining. Results
were expressed as % positively stained area per 2006 field. (*p,0.05
compared with Veh; **p,0.05 compared with LPS/Veh).
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PLoS ONE | www.plosone.org8 May 2010 | Volume 5 | Issue 5 | e10656
Figure 5. Distamycin A disrupts binding of an inducible protein-DNA complex containing NF-kB to an AT-rich region of the P-
selectin promoter. A. As in Fig. 3B, lung tissue was harvested from wild type mice two hours following treatment with Vehicle (Veh), LPS/Vehicle,
or LPS/Dist A (Dist), then subjected to RNA extraction. The same blot from Fig. 3B was hybridized with a radiolabeled probe for HMGA1 (and an 18S
HMGA1 and Inflammation
PLoS ONE | www.plosone.org9 May 2010 | Volume 5 | Issue 5 | e10656
addition of Dist A (in the presence or absence of TNF-a, lanes 5,7
in lower panel) inhibited in vivo HMGA1-DNA binding to the
designated AT-rich region of the P-selectin promoter (35.064.9%
reduction in binding for TNF-a/Dist compared with TNF-a/
This study reports three important new findings. First, we
present data supporting a novel anti-inflammatory strategy in vivo
through using a minor groove binding drug to specifically interfere
with DNA-protein binding in a targeted manner. Second, we
demonstrate an important role for the architectural transcription
factor HMGA1 in facilitating full induction of P-selectin promoter
transactivation and inflammatory-induced gene expression. Third,
we demonstrate that using transcriptional regulation to target
select, similarly regulated inducible genes with common promoter
motifs can be effective in improving outcomes in murine models of
critical illness. Furthermore, our data supports the intriguing
concept that minor groove binders can serve as an important in
vivo tool to dissect molecular mechanisms of inflammatory disease
Our previous work employed MGBs in vitro [25,26,33,42] and in
vivo  to confirm an important role for TF-binding to AT-rich
DNA regions in transactivation of the NOS2 promoter and in
attenuating mortality and hypotension during murine endotox-
emia. Numerous genes critical for the inflammatory cascade share
similar promoter regulatory regions with NOS2, and we therefore
hypothesized that MGBs would attenuate the inflammatory
response through similarly interfering with TF-binding to AT-
rich DNA regions of promoters of genes critical for the
inflammatory response. Recruitment of inflammatory cells to the
tissue occurs via a complicated cascade of events, each step of
which is highly regulated . Leukocytes traversing the
vasculature at rapid speed are lured to the activated endothelium
through initial tethering and rolling mediated predominantly by
members of the selectin family of adhesion molecules (P-selectin,
E-selectin, and L-selectin). Leukocytes are then activated with
subsequent firm adhesion to the endothelial surface facilitated in
large part through interaction of immunoglobulin family members
(on endothelial cell surface) with integrins (on leukocytes).
Transmigration of leukocytes across the endothelial surface and
into the tissue is likely mediated by a gradient of chemoattractants.
Given the role of MGBs in attenuating inducible gene
expression  and the observed reduction of rolling fraction
and sticking efficiency of leukocytes to the endothelial surface
(Figure 2), we focused our studies on inducibly expressed genes
previously demonstrated to play a role in the early tethering and
rolling phases of leukocyte-endothelial surfaces. Therefore, we
examined the effects of MGBs on P-selectin and E-selectin, both of
which are inducibly expressed in endothelial cells (, Figure 3).
Of note, L-selectin is constitutively expressed on leukocytes .
While numerous members of the adhesion molecule families
provide a contributory role toward leukocyte sticking and rolling
[54,56–61], studies of knockout animals have revealed that P-
selectin plays the most critical and pronounced role in early
leukocyte tethering and rolling [54,62]. Therefore, the fact that
induction of P-selectin can be selectively attenuated in this model
(Figure 3) represents an important example of the potential in vivo
benefits of exquisite transcriptional control. Similar to the
observed effects of MGBs on NOS2 in this model ,
inflammatory cell recruitment and P-selectin induction was
decreased but not entirely abolished with MGB treatment
(Figure 3). Interestingly, other investigators have reported that
NOS2 does not play a role in regulating expression of endothelial
adhesion molecules, suggesting that our findings regarding P-
selectin are independent of the regulation of NOS2 expression by
Dist A in this model . Furthermore, P-selectin exhibits
constitutive as well as inducible expression within endothelial cells
[11,12] such that the effect of leukocyte-endothelial interactions
attributable to P-selectin is reduced but not eliminated with
MGBs. Thus, the ability to selectively control gene transcription
through interfering with TF-DNA binding presents the potential
for a ‘‘titratable’’ anti-inflammatory effect, versus the ‘‘all or none’’
effect of more traditional anti-inflammatory approaches.
Interestingly, P-selectin is fairly unique as an adhesion molecule,
as it is expressed not only in endothelial cells, but also in platelets,
and roles of P-selectin in different locations has been a matter of
recent debate. While numerous studies reported an important role
for endothelial P-selectin expression in lung and liver inflamma-
tion and physiologic injury during endotoxemia [49–51], more
recently, interest has arisen in the importance of platelet P-selectin
expression in development of acid-induced lung injury . While
we cannot fully exclude the role of platelet P-selectin in our studies,
examination of histologic sections (Figure 4) and our studies in
endothelial cells in vitro (Figures 5–6) support a significant
contribution of endothelial P-selectin to our observations.
Moreover, a recent study reported that P-selectin glycoprotein-
ligand-1 regulates lung neutrophil recruitment independently of
circulating platelets in a murine model of abdominal sepsis (cecal
ligation and puncture model ). In aggregate, our findings in
conjunction with those in the literature support the intriguing
possibility that mechanisms of tissue neutrophil recruitment during
indirect lung injury (e.g., systemic endotoxin, abdominal sepsis) are
distinct from those that predominate during direct injury. Tissue
neutrophil recruitment during indirect lung injury might rely more
probe as loading control). This experiment was repeated two separate times. B. Nuclear extracts from BAEC cells (Lanes 1–7) and primary murine lung
endothelial cells (MLEC, Lanes 8–12) (‘‘Nuc Ext’’) with (Lanes 3–7, 9–12) or without (Lanes 2,8) TNF-a stimulation were subjected to electrophoretic
mobility shift assays (EMSA) using a radiolabeled probe spanning the AT-rich region of the P-selectin promoter (basepairs 2542 to 2521). Lane 1
represents the radiolabeled probe without addition of nuclear extract. TNF-a-treated nuclear extract was additionally incubated and electrophoresed
with the radiolabeled probe and vehicle (V, lanes 3 and 10 (or in the presence of TNF-a without vehicle, Lane 9)) or increasing concentrations of Dist A
(D1=10 mM, D2=20 mM, Lanes 4–5, 11–12) as well as with an identical competitor (IC, Lane 6) and a non-identical competitor (NIC, Lane 7).
(* represents the inducible, specific complex seen following TNF-a treatment; ‘‘R’’ represents disruption of the TNF-a-inducible complex following
addition of Dist A (Lanes 4–5 compared with Lane 3 and Lanes 11–12 compared with Lane 9–10). All of the binding studies were repeated at least two
separate times. C. Nuclear extracts from BAEC cells (‘‘Nuc Ext’’) with (Lanes 3–7) or without (Lane 2) TNF-a stimulation were subjected to
electrophoretic mobility shift assays (EMSA) using a radiolabeled probe spanning the AT-rich region of the P-selectin promoter (basepairs 2542 to
2521). Lane 1 represents the radiolabeled probe without addition of nuclear extract. TNF-a-treated nuclear extract was additionally incubated and
electrophoresed with the radiolabeled probe and antibodies to the NF-kB family members p50 and p65 (lanes 4–5), or unrelated and control
antibodies (Ets-1 and IgG control respectively, lanes 6–7). (* represents the inducible, specific complex seen following TNF-a treatment; ‘‘r’’
represents supershifted band/disruption of the TNF-a-inducible complex following addition of p50 and p65 antibodies (Lanes 4-5 compared with
Lanes 6–7). All of the binding studies were repeated at least two separate times.
HMGA1 and Inflammation
PLoS ONE | www.plosone.org10 May 2010 | Volume 5 | Issue 5 | e10656
Figure 6. HMGA1 binds to the P-selectin promoter and is critical for full induction of P-selectin promoter activity. A. BAEC cells were
transiently transfected with a P-selectin promoter-reporter construct with the addition of a blank expression vector, an expression vector for HMGA1,
and/or expression vectors for NF-kB family members (p50/p65). Transfected cells were harvested and assessed for luciferase activity (normalized for
b-galactosidase content). Results are expressed as fold-change in luciferase activity relative to transfection with the P-selectin promoter and a blank
expression vector. B. BAEC cells were transiently transfected with a P-selectin promoter-reporter construct and increasing concentrations of a vector
expressing a dominant-negative form of HMGA1 (DN-HMGA1). Transfected cells were stimulated with TNF-a, then harvested and assessed for
luciferase activity (normalized for b-galactosidase content). Results are expressed for each transfection condition as fold-change in luciferase activity
as a result of TNF-a stimulation. (*p,0.05 for 0.5 mg of DN-HMGA1 as compared with empty vector control; **p,0.05 for 1.0 mg of DN-HMGA1 as
compared with empty vector control). This experiment was repeated three separate times, with each condition performed in triplicate. C. An
electrophoretic mobility shift assay (EMSA) was performed using the HMGA1 peptide and a radiolabeled probe spanning the AT-rich region of the P-
selectin promoter (basepairs 2542 to 2521) without (Lane 2) or with Dist A (10 mM, Lane 3). Lane 1 represents the radiolabeled probe in the absence
of incubation with protein. (* represents the HMGA1-DNA complex in Lane 2 which is diminished in intensity following addition of Dist A in Lane 3).
HMGA1 and Inflammation
PLoS ONE | www.plosone.org11May 2010 | Volume 5 | Issue 5 | e10656
heavily on endothelial P-selectin expression, while platelet P-
selectin expression may play a more prominent role during direct
Our results indicate that MGBs can serve as a useful tool to
probe the functional effects of targeted DNA sequences in
complicated biological systems in vivo. Through observing the
effects of MGBs in inhibiting HMGA1-binding to a targeted AT-
rich region of the P-selectin promoter and in demonstrating the
effects of the dominant-negative HMGA1 construct in attenuating
induction of the P-selectin promoter by TNF-a, we were able to
derive an important role for HMGA1 in regulating P-selectin
promoter induction (Figures 5–6). We acknowledge that exami-
nation of the effect of MGBs on P-selectin induction in the
presence of HMGA1 knockdown would provide more direct
evidence that HMGA1 expression is essential for the observed
effects of DistA. However, these experiments were not technically
feasible, given the interference of the siRNA reagents with the
Other investigators have elegantly demonstrated in Drosophila
that targeted minor-groove binding drugs can be fed to flies that
interfere with binding of the Drosophila HMGA1 orthologue
(termed D1) to specific AT-rich DNA sequences and result in
specific gain- and loss-of-function phenotypes [66–69]. In these
studies, small cell-permeable molecules were synthesized based
upon the existing known structure of Distamycin A and were
designed with the goal of developing improved tools to elucidate
the role of architectural DNA regions in biology in model systems
. In recent years, there has been increasing interest in
development of derivatives of MGBs as human chemotherapeutics
to allow targeted delivery of DNA-modifying agents [69,70].
Furthermore, improvements in techniques to elucidate molecular
structure has led to a growing literature on detailed characteriza-
tion of MGB-DNA binding as well as on development of novel
compounds with optimized DNA-binding and functional proper-
ties [71–75]. Our data supports the premise that novel small
molecules interfering in a targeted way with sequence- and
conformation-specific DNA binding can be studied at the
molecular and physiologic level in higher order organisms
subjected to models of human disease. Such approaches hold
promise for development of novel treatment strategies for critical
illness. To our knowledge, the present study and our prior work
[33,43] represent the first in vivo applications of MGBs to murine
models of critical illness.
In summary, we now demonstrate that MGBs can interfere in a
targeted manner with HMGA1 binding to the P-selectin promoter
in vivo, resulting in attenuated P-selectin induction and decreased
lung and liver inflammation during murine endotoxemia. These
findings, in combination with our previous data showing
improvement in mortality and hypotension during murine
endotoxemia attributed to attenuated NOS2 induction ,
supports the interesting possibility that there exist select genes
regulated by common promoter motifs that can be advantageously
regulated to improve outcomes from critical illness. With a
growing appreciation of conserved regulatory motifs throughout
the human genome and with increasing ability to catalogue this
data , there exists a real possibility of molecularly targeted
treatment strategies that can be applied in individualized ways to
complex human disease. We acknowledge that MGBs may have
other effects on an organism that remain to be characterized.
However, our work represents implementation of MGBs as a
molecular tool to derive in vivo biological characterization of
critical illness that ultimately can be applied to the development of
novel therapeutics in a field where effective treatment approaches
are desperately needed.
The authors are grateful to Bonna Ith for technical assistance.
Conceived and designed the experiments: RMB MDL UvA MP.
Performed the experiments: RMB SLG DFR AAM GC CH SWC.
Analyzed the data: RMB SLG DFR AAM MDL GC CH SWC RR UvA
MP. Contributed reagents/materials/analysis tools: RMB MDL RR UvA
MP. Wrote the paper: RMB.
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