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INFLAMMATION, CYTOKINES, AND TEMPERATURE REGULATION
1Shriners Hospitals for Children and Department of Surgery, and 2Department of Human Genetics and Biological Chemistry, University of Texas Medical Branch, Galveston, Texas 77550
Submitted 24 January 2003 ; accepted in final form 9 June 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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B transcription factors. Data were consistent with our
hypothesis that the beneficial effects of IGF-I gene transfer on burned rats
act in part via activation protein-1 and NF-B transcriptional
regulation and the concordance between the results obtained with
antiapoptotic, as opposed to the proapoptotic, sequences as well as the
corresponding changes in measures of cell death via Bax and caspase-3
mechanisms.
nuclear factor-B; thermal injury; activation protein-1; caspase-3; Bax
Delayed cell death displays characteristic molecular changes that include
increased activation of caspase enzymes and shifts in the levels and
intracellular occupancy of the Bcl-2 family of proteins
(6). For example, several
death-promoting pathways converge on increased Bax expression
(26). The transcription
factors activation protein-1 (AP-1) and NF-B have been shown to be key
regulators of cell death and inflammatory cascades.
Members of the AP-1 transcription factor family have been shown to play an important role in cell proliferation, differentiation, and survival (1). The AP-1 complex recognizes a set of specific DNA sequences (12-O-tetradecanoylphorbol-13-acetate responsive elements), present in many enhancer or promoter region of genes (1). Changes in the relative levels of the different members of the AP-1 family or their state of phosphorylation serve to regulate cell death vs. cellular proliferation (1). In some cell types, repression of AP-1 activity has been shown to trigger apoptosis (11).
The NF-B family of transcription factors regulates genes mediating
inflammation, responses to infection, oxidative stress, and the aftermath of
ambient necrotic events (3).
NF-B is made up of five structurally related protein subunits: p50,
p52, p65/RelA, c-Rel, and RelB, which form a variety of homo- and heterodimers
in multiple tissues (24). All
five NF-B/Rel proteins share a 300-amino acid region, the Rel homology
domain, which is the structural basis for dimerization, DNA binding, and
nuclear localization (20).
NF-B activation can be measured by determining levels of binding of
nuclear extracts to oligonucleotides bearing the different NF-B-binding
consensus DNA sequences present in specific gene promoters of interest
identified by immunodepletion/supershift assays. NF-B dimers bind
target gene regulatory regions through a wide variety of binding sites, which
generally match a 5'-GGGRNTY(C/T)C-3' consensus (R = A or G, Y = C
or T, N = any nucleotide). NF-B achieves target gene specificity, in
part, through preferential binding of different subunit combinations to the
different DNA consensus-binding sequences
(10,
23,
24,
35). The inhibition of total
NF-B binding to DNA consensus sequences present in the IgG-B
promoter have been shown to increase apoptotic cell death
(32,
33). However, the consequences
of more selective interventions in the interactions between specific
NF-B protein dimers and gene-specific DNA consensus sequences present
in the promoter of genes have not been determined; the expression of these DNA
consensus sequences is known to be altered by stress, and they are known to
have significant effects on apoptotic outcomes.
In the present study, we express transient increased levels of IGF-I protein in rats exposed to thermal trauma via liposomal gene transfer as described elsewhere (17) in an effort to define the transcriptional events that occur after IGF-I delivery at the site of injury. We previously established the optimal delivery parameters and the attenuating role of IGF-I on inflammatory cytokine production (16, 29).
Here, we demonstrate the beneficial effects of IGF-I gene transfer and its
action, partly via amelioration of burn-induced inflammatory responses that
mediate cell death, such as caspase-3 activity and Bax expression. We also
show that IGF-I gene transfer induces selective stimulation of AP-1
DNA-binding activity and the activation of antiapoptotic, but not
inflammatory, NF-B transcription factors.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Anesthesia was induced by intraperitoneal injection of pentobarbital sodium (50-90 mg/kg) and buprenorphine tartrate (0.1-1.0 mg/kg). Rats were shaved and subjected to 40% total body surface area full-thickness scald burn on the dorsum and ventrum as previously described (12). Burned rats were resuscitated with lactated Ringer solution (60 ml/kg) and randomized into treatment or control groups. All the rats were placed on a warming pad after injury and, once awake, were returned to their cages. Sham-treated rats received anesthesia and equivalent handling without a scald burn. Six rats in each group were killed 240 h after burn trauma.
Burned rats received 0.2 ml of the liposome solution [2.2 µg of DNA in
20 µl of 1,2-dimyristyloxypropyl-3-dimethyly-hydroxyl ethyl ammonium
bromide (DMRIE-C) in 180 µl of saline] or saline at two sites, each 1 cm
from the burn wound margin. Rats were killed after 240 h by decapitation. Skin
samples (
1 cm2) were harvested from the wound border at the
injection site and from corresponding sites in control animals. Samples were
immediately frozen in liquid nitrogen and stored at -70°C for
analysis.
The IGF-I-cDNA treatment solution consisted of 10 µl of liposome solution in 180 µl of saline containing 2.2 µg of an IGF-I cDNA construct and 0.2 µg of cDNA of the reporter gene LacZ. The liposome solution is made up of the cationic lipid DMRIE-C (DMRIE-C reagent, GIBCO BRL Life Technologies, Rockville, MD). All lipid-DNA complexes were freshly prepared 2 h before injection. The IGF-I cDNA construct consisted of a cytomegalovirus-driven IGF-I cDNA plasmid prepared at the University of Texas Medical Branch Sealy Center for Molecular Science Recombinant DNA Core Facility. Burn controls received an equal amount of saline injected subcutaneously.
This study was approved by the Animal Care and Use Committee of the University of Texas Medical Branch and followed the guidelines established by the National Research Council.
EMSA. Nuclear protein extracts from the frozen skin samples were
prepared by the method of Dignam et al.
(7). Nuclear extracts were
stored at -70°C until used. Protein concentrations of extracts were
measured using the bicinchoninic acid assay (Pierce). Oligonucleotides
encompassing the IgG-B enhancer sequence (GGGACTTTCC) and the
bcl-x gene NF-B/CS4 sequence (GGGGGTCTCC) and AP-1 consensus
oligonucleotide (5'-CGCTTGATGACTCAGCCGGAA-3') were used as probes,
labeled at the 5' end with [
-32P]ATP and T4
polynucleotide kinase, and purified. Binding reaction buffer consisted of 10
mM HEPES, pH 7.9, 60 mM KCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, 0.2 mM PMSF,
0.5 µg of poly(dI-dC), 12% glycerol, 10 µg of nuclear extract, and
20,000 cpm end-labeled oligomer probe. When antibodies were used in EMSA for
the immunodepletion/supershift study, nuclear extracts were incubated with the
different antibodies for 30 min at 4°C before the addition of the
poly(dI-dC). All antibodies except NF-B p52 (catalog no. 06-413,
Upstate Biotechnology) were purchased from Santa Cruz Biotechnology (SC-6955x
for NF-B c-Rel, SC-372x for NF-B p65, SC-7178x for NF-B
p50, and SC-226x for NF-B Rel-B) and then loaded onto a 6%
polyacrylamide-0.5x Tris base-boric acid-EDTA gel and run for 3 h at 120
V. After electrophoresis, gels were dried and exposed to intensifying screens
for phosphor-imaging analysis (Molecular Dynamics) and then to Kodak X-OMAT
film.
Immunoblotting. Total protein extracts from skin (20 µg) were added to sample buffer and separated on a 10% SDS-polyacrylamide gel under reducing conditions and transferred to nitrocellulose membranes (Hybond-C, Amersham Pharmacia Biotech) in a semidry blotting chamber. After nonspecific binding sites were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (Sigma, St. Louis, MO), blots were incubated in a 1:3,000 dilution of anti-Bax rabbit polyclonal antibody (Santa Cruz Biotechnology) for 2 h at room temperature. After three to five washes, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (final concentration 1:3,000) for 90 min at room temperature. Bound antibodies were detected with enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The intensities of the bands were quantitated using densitometry.
Colorimetric determination of caspase activity. Caspase-3 activity was determined by a colorimetric assay (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. Briefly, tissue extracts of 100-200 µg of total protein were incubated with 5 µl of a caspase-3-specific peptide conjugated with p-nitroaniline (IETD-pNA) in reaction buffer at 37°C for 2 h; then light absorption was read at a wavelength of 405 nm together with controls. Results were compared between groups.
Statistical analysis. Values are means ± SE. Unpaired t-tests were used for analysis of differences between various treatments. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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NF-B IgG-binding activity in rat burn skin. To
determine the effects of IGF-I gene transfer on the inflammatory contribution
of NF-B transcriptional activation to burn trauma, we measured
NF-B DNA-binding activity by EMSA using the DNA consensus sequence in
the IgG-B promoter, known to be relevant to transcriptional regulation
of inflammatory signal cascade molecules. We determined the
NF-B-binding activity in the nuclear extracts of the rat skin after 10
days in all three groups by EMSA. IGF-I-treated burned rats showed decreased
NF-B-binding activity compared with burned rats without IGF-I treatment
(Fig. 3).
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NF-B binding to Bcl-XL
promoter in rat burn skin with and without IGF-I treatment. To
determine the noninflammatory NF-B contribution, we measured the
effects of IGF-I gene transfer on NF-B binding to Bcl-x promoter using
NF-B DNA consensus sequences. The NF-B-binding activity to
Bcl-XL promoter in nuclear extracts of the rat skin 10 days after
injury and IGF-I gene transfer showed significant increases compared with
burned rats without IGF-I treatment (EMSA;
Fig. 4).
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Composition of NF-B in rat skin 10 days after burn
injury with and without IGF-I treatment. With the purpose of determining
the composition of the NF-B transcription factor DNA-binding activity
in rat skin after burn injury and IGF-I treatment, we examined the composition
of NF-B-32P-oligonucleotides-protein complexes. We added
antibodies specific to the different subunits of NF-B before carrying
out EMSA so as to interfere with DNA-binding events involving specific
subunits of NF-B. The results of the assays
(Fig. 5) reflected the relative
contributions of the subunits to the NF-B-binding activity measured as
a supershift or reduction in binding activity. For example, as shown in
Fig. 5, addition of antibodies
to p50 induced a significant reduction in the amount of NF-B DNA
binding detected by EMSA, suggesting that the p50 subunit was most likely
participating in NF-B DNA-binding activity. There were changes in the
degree of DNA-binding activity through the p50 subunit
(Fig. 6). There was a prominent
increase in p50-induced DNA-binding activity of NF-B in IGF-I-treated
burned rats compared with untreated burned rats.
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AP-1 DNA-binding activity in rat burn skin. We measured AP-1 DNA-binding activity in sham-treated and burn-injured rat skin after 10 days with and without IGF-I treatment by EMSA. Although AP-1-binding activity was significantly reduced in burn-injured skin, its activity increased in IGF-I-compared with sham-treated rats (Fig. 7).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Wound healing is a dynamic interactive and complex process involving soluble mediators, blood cells, extracellular matrix, and parenchymal cells (28). Different cell types synthesize and release different mediators that modulate and stimulate the cascade of wound healing. Thus the response to thermal trauma consists of cascades that contribute to pathology and loss of function as well as to recovery processes. Mechanisms of recovery consist of different signaling and effector pathways working in concert with overlapping and cross-reacting elements, a redundant feature. However, growth factors have been suggested to influence and modulate epidermal and dermal cell survival and regeneration (27, 28). Among the growth factors that have been shown to improve wound healing is IGF (36, 37). Because of enzymes, proteases, and lack of receptors, the local application of growth factors as proteins has been shown to be ineffective (28), an advantage of our gene transfer approach. Although it is known that systemic IGF-I injections are beneficial after burn trauma, the associated increases in IGF-I systemic levels have drastic toxic side effects.
In burn trauma, this cascade of events is not only a locally defined process at the wound site, but it also extends its effects via release of inflammatory mediators at a systemic level. Given that the demonstrated beneficial effects of growth factors, such as recombinant growth hormone protein treatment, act in part through IGF-I effects at the periphery, peripheral treatment of burn trauma is warranted. We have demonstrated that, in transient gene transfer of an IGF-I construct under control of a cytomegalo-virus promoter, there is an acceleration of reepithelialization rates, most significantly during the 1st mo after burn trauma (17).
Nonviral gene therapy has several advantages over viral gene therapy. Nonviral delivery systems typically result in robust, prompt, and transient localized gene expression without triggering confounding immunologic responses or tachyphylaxia (16, 34). We have determined optimal liposome-to-DNA ratios, biodistribution, and feasibility of nonviral liposomal cDNA injections into the skin (16, 34). We showed that, after the injection of liposome-cDNA complexes, the transferred gene was transcribed into mRNA and translated into the protein (15), which was more potent in a dose-dependent fashion than the same factor administered as a recombinant protein at a 100-fold-higher concentration (17, 34). As part of these studies, we thoroughly characterized the effects of different liposomal preparations on burned rats and also on control rats and showed no differences in the reepithelialization between saline-injected and liposome-treated animals (18, 34).
IGF-I, a 7.7-kDa, single-chain, 70-amino acid polypeptide with sequence homology to proinsulin, improves metabolic rate, gut mucosal function, and protein loss after a burn injury (2, 13, 14, 31). IGF-I is bound and transported with one of its six binding proteins (2) and mediates the actions of growth hormone in the hypermetabolic state by attenuating lean body mass loss, improving the immune response, attenuating the acute-phase response, and enhancing wound healing (5, 15, 17, 22). Fibroblasts and keratinocytes have IGF-I receptors, which probably mediate IGF-I stimulation of mitogenicity and proliferative activity (21). In experimental and clinical studies, the great potential of IGF-I to improve wound healing has been shown (21, 30). Our group previously showed a significant acceleration of morphological structure, such as collagen bundle formation, immunohistochemically during wound healing, which is important in terms of the remodeling taking place beneath the burn site as a consequence of IGF-I therapy (19).
Inflammation is a distinct phase in wound healing, releasing biologically active substances, which stimulate the migration of host cells to the injury site (4). Reduced or delayed inflammation may delay the overall healing process and may predispose tissues to infection by permitting the early establishment of invading microorganisms before the recruitment of host cells. However, inflammation also stimulates the synthesis of the cytokines and growth factors, which, in turn, alter cellular commitments to apoptosis, cellular metabolic rates with outcomes that include the perturbation of growth and developmental patterns in young children (9, 25, 29).
It is known that the NF-B transcription factor activates gene
expression of antiapoptotic genes, such as the Bcl-2, Bcl-x family,
specifically via c-Rel/p50 NF-B binding to consensus sequence on
antiapoptotic genes (24,
32), and that prolonged
activation of the p65/p50 NF-B results in proapoptotic gene expression
and delayed cell death (10).
Not surprisingly, transient effects of burn trauma on NF-B activation
cease 10 days after burn injury. However, we found IGF-I gene transfer-induced
increases in binding of NF-B to Bclx-specific NF-B-binding DNA
consensus sequences, such as those found in the Bcl-x promoter, 10 days after
burn trauma (10,
24). Our previous study, using
promoter constructs for the bcl-x gene, which has NF-B
consensus-binding sites, in a reporter gene assay showed similar observations
(10).
Using RNase protection assays, we previously showed a selective stimulation
of the TNF-
and IL-1
cytokines in skin samples over time in
IGF-I-treated burn-injured rats compared with untreated burn-injured rats at
10 days (29), consistent with
our hypothesis that the mechanism of action of the IGF gene transfer
therapeutic effects is via an amelioration of local inflammation triggered by
the burn trauma event. Given that IGF-I action via IGF-I receptors involves
triggering of the Akt signaling pathway, directly involved in NF-B
transcription factor regulation of inflammatory responses, our finding is
compatible with IGF-I gene transfer on NF-B activation of pro- and
antiapoptotic gene promoter sequences associated with inflammatory responses.
It is consistent with our hypothesis that the beneficial effects of IGF-I gene
transfer on burned rats act in part via AP-1 and NF-B transcriptional
regulation. Most exciting is the concordance between the results obtained with
anti- and proapoptotic sequences as well as the corresponding changes in
measures of cell death via Bax and caspase-3 mechanisms. These would suggest
that local IGF-I levels could have profound consequences on the triggering of
inflammatory signal cascades. The role of this intervention on other factors
acting later in the wound-healing process remains to be determined.
This broad array of responses is consistent with the improved outcomes reported by us and others using multiple local injections every week (18, 25). Such an approach would be consistent with the surgical procedures used in the clinical care of burns. Understanding the signal transduction pathways acting between the periphery and the liver in response to trauma and effective therapeutic interventions as assayed deserves further investigation. Although some validation is provided here in terms of selective regulation of certain gene products, intervention in IGF-binding protein effects on IGF-I by the addition of exogenous IGF-binding protein, increasing apoptotic outcomes via Bax transfections, Bcl-x antisense treatment, or induction of hypoglycemia as a way of compromising energy metabolism would yield clues to the regression from the two transcription factor activities. This in turn may help identify the regulatory features triggered by thermal trauma and the beneficial effects of IGF-I gene transfer.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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)
subunit of the high-molecular-weight insulin-like growth factor-binding
protein complex. J Clin Endocrinol Metab
70: 1347-1353,
1990.[Abstract]
B in preventing
TNF--induced cell death. Science
274: 782-784,
1996.1-acid glycoprotein and
1-antitrypsin correlates with sensitivity to thermal injury.
Surgery 119:
664-673, 1996.[ISI][Medline]
B binding sites in the murine bcl-x promoter. J
Neurochem 75:
1377-1389, 2000.[ISI][Medline]
B/rel transcription factor and I
B inhibitor system. Curr Opin Cell Biol
5: 477-487,
1993.[Medline]
B subunits determine the specificity of
transcriptional activation. Proc Natl Acad Sci USA
89: 1529-1533,
1992.B regulation of bcl-x gene expression in
hippocampus and basal forebrain in response to hypoxia. J Neurosci
Res 64: 223-234,
2001.[ISI][Medline]
B.
Neuroreport 9:
489-493, 1998.[ISI][Medline]
B
(NF-B) activity induces nerve growth factor-resistant apoptosis in PC12
cells. J Neurosci Res 47:
155-162, 1997.[ISI][Medline]
B/p49 is a negative regulatory factor in nerve growth factor-induced
choline acetyltransferase promoter activity in PC12 cells. J
Neurochem 75:
2241-2251, 2000.[ISI][Medline]
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