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* From the Division of Pulmonary and Critical Care Medicine (Drs. Otterbein, Chin, and Petrache), The Johns Hopkins University School of Medicine, Baltimore, MD; Department of Molecular Genetics (Dr. Alam), Alton Ochsner Medical Foundation and Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, LA; Section of Pulmonary and Critical Care Medicine (Drs. Otterbein, Lee, Chin, and Choi), Yale University School of Medicine, New Haven, CT, and Connecticut VACHS, West Haven, CT; and Division of Pulmonary and Critical Care Medicine (Dr. Camhi), University of Arizona, Tucson, AZ.
Correspondence to: Augustine M.K. Choi, MD, Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, 333 Cedar St, LCI 105, New Haven, CT 06520; e-mail: augustine.choi{at}yale.edu
Heme oxygenase (HO) catalyzes the first and rate-limiting step in the oxidative degradation of heme to bilirubin1 2 (Fig 1) . The binding of HO with the heme molecule leads to the cleavage of a meso carbon bond and results in the production of biliverdin, which is subsequently converted to bilirubin by biliverdin reductase.1 2 Three isoforms of HO exist; HO-1 is highly inducible, while HO-2 and HO-3 are constitutively expressed.1 2 Although heme is the major substrate of HO-1, a variety of nonheme products, including heavy metals, cytokines, hormones, endotoxin, and heat shock, are also strong inducers of HO-1 expression.1 In addition, HO-1 is highly induced by a variety of agents causing oxidative stress, including hydrogen peroxide, glutathione depletors, ultraviolet irradiation, endotoxin, and hyperoxia.1 3 4 This diversity of HO-1 inducers has provided further support for the speculation that HO-1, besides its role in heme degradation, may also serve to play a vital function in maintaining cellular homeostasis. We will review some of the experimental data that support the evolving paradigm that the stress inducible gene HO-1 plays an important functional role in the lung and host's defense against oxidant-induced lung injury.
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Regulation
Using hyperoxia as a model of oxidant-induced lung injury in the
rat, we have examined the regulation of HO-1 expression in
vivo and in vitro. We have demonstrated marked increase
of HO-1 messenger RNA (mRNA) and protein levels in the rat lungs after
hyperoxia.4
Immunohistochemical studies of the rat lung
after hyperoxia showed increased HO-1 expression in a variety of cell
types, including the bronchoalveolar epithelium, interstitial cells,
and inflammatory cells.4
We have also observed increased
HO-1 gene expression in vitro after hyperoxia in various
cultured cells, including epithelial cells, fibroblasts, macrophages,
and smooth muscle cells.4
Increased HO-1 mRNA expression
correlated with increased HO-1 protein in vitro, and
resulted from increased gene transcription and not from increased mRNA
stability.4
We have also demonstrated that the
transcriptional activation of the HO-1 gene by hyperoxia requires
cooperation between the HO-1 proximal promoter and an enhancer fragment
called SX2 located 4 kb upstream from its transcription
site.4
Function
We hypothesized that HO-1 induction serves to protect cells
against hyperoxia. We successfully stably transfected human pulmonary
epithelial cells (A549 cells) with HO-1 complementary DNA (cDNA) and
demonstrated that these cells exhibited marked induction of HO-1 mRNA.
This increased HO-1 mRNA correlated with increased HO-1 enzyme
activity.5
Cells that overexpress HO-1 exhibited a marked
decrease in cell growth compared with wild-type A549 cells or A549
cells transfected with control DNA.5
This decreased cell
growth was associated with an increased number of cells in Go/G1 phase
during the proliferative exponential phase and decreased entry into the
S phase determined by flow cytometric analysis of propidium
iodide-stained cells and pulse experiments with bromodeoxyuridine.
Furthermore, the A549 cells overexpressing HO-1 accumulated at the G2/M
phase and failed to progress through the cell cycle with serum
stimulation, whereas the A549 cells transfected with control DNA
exhibited normal cell cycle progression.5
Interestingly,
the A549 cells overexpressing HO-1 also exhibited marked resistance to
hyperoxic oxidant insult. Tin protoporphyrin, a selective inhibitor of
HO, reversed the growth arrest and ablated the increased survival
against hyperoxia observed in the HO-1 overexpressing A549
cells.5
Taken together, our data suggest that
overexpression of HO-1 results in cell growth arrest, which may
facilitate cellular protection against nonheme-mediated oxidant insult
such as hyperoxia. A recent report also demonstrated that HO-1
induction in vivo protected against hyperoxia-induced lung
injury.6
We examined whether exogenous administration of HO-1 by gene transfer could confer protection against oxidant-induced tissue injury. We first demonstrated the feasibility of overexpressing HO-1 in the lung via gene transfer. A 1.0-kilobase pair Xho I/Hind III fragment of the rat HO-1 cDNA clone containing the entire coding region was cloned into plasmid pAC-CMVpLpA and recombinant adenoviruses containing the rat HO-1 cDNA fragment, Ad5-HO-1, were generated by homologous recombination. The recombinant adenovirus containing the Escherichia coli ß-galactosidase gene, AdV-ßGal, served as vector controls. We administered Ad5-HO-1 (109 pfu) intratracheally to Sprague Dawley rats and examined HO-1 expression in rat lungs. Increased HO-1 protein expression was detected diffusely in the bronchiolar epithelium of rats receiving Ad5-HO-1 as assessed by immunohistochemical studies. We then sought to examine whether ectopic expression of HO-1 could confer protection against hyperoxia-induced lung injury. Rats receiving Ad5-HO-1 prior to exposure to hyperoxia (> 99% O2) exhibited marked reduction in lung injury as assessed by volume of pleural effusion (> 90% reduction) and histologic analyses (significant reduction of edema, hemorrhage, and inflammation) in response to hyperoxia. In addition to attenuation of lung tissue injury, rats receiving Ad5-HO-1 also exhibited increased survivability against hyperoxic stress when compared with rats receiving a recombinant adenovirus encoding the E coli ß-galactosidase gene, AdV-ßGal.
Role of HO-1 in Endotoxin-Induced Lung Injury
Regulation
Lipopolysaccharide (LPS) when administered in vivo
produces pathophysiologic changes similar to those seen in human ARDS.
The pathogenesis of these changes is in part mediated by oxidative
stress. We have demonstrated that LPS induces high mRNA levels of the
stress-inducible gene HO-1 in the rat lung.7
Increased
HO-1 mRNA levels correlate with increased HO-1 protein and enzyme
activity. Immunohistochemical analyses of lung tissues from rats
treated with LPS reveal abundant HO-1 expression in inflammatory and
bronchoalveolar epithelial cells.7
We have further
examined the molecular regulation of HO-1 gene expression following
exposure of RAW 264.7 macrophage cells to LPS in vitro.
These cells respond to LPS with increased HO-1 mRNA expression and HO-1
gene transcription. Transcriptional activation of the mouse HO-1 gene
by LPS is mediated by a 5' distal enhancer fragment located
approximately 4-kilobase pair upstream from the transcription site.
Electrophoretic mobility shift assays show increased activator
protein-1 (AP-1) binding activity in RAW 264.7 cells after LPS
treatment. Mutation of the AP-1 binding site in this enhancer fragment
completely abolishes HO-1 gene activation while mutation of
CCAAT/enhancer-binding protein binding site exerts negligible effect,
suggesting that the AP-1 family of transcription factors plays a
critical role in regulating HO-1 gene activation following LPS
treatment.7
Function
In a rat model of LPS endotoxin-induced lung injury and sepsis, we
have demonstrated that the lung responds to LPS by expressing high
levels of HO-1 mRNA and enzyme activity. We hypothesized that this HO-1
induction could play a critical role in the lung's defense against
LPS. Pretreatment of rats with hemoglobin (Hb), a potent inducer of
HO-1, resulted in HO-1 induction and more importantly provided complete
protection against subsequent lethal endotoxemia.8
Hb-treated rats maintained normal mean arterial BP while control rats
experienced complete cardiovascular collapse after a lethal dose of
LPS. Hepatic and renal functions, peripheral WBC count, serum lactate
dehydrogenase level, and phosphate level also remained normal after LPS
in Hb-treated rats. Hb in a dose conferring 100% survival after a
lethal dose of LPS attenuated LPS-induced neutrophil alveolitis
(>90%) and tumor necrosis factor-
levels (>40%).9
Tin protoporphyrin, a competitive inhibitor of HO, blocked this
protective effect of Hb and rendered the rats more susceptible to
LPS.8
The precise mechanism by which HO-1 confers protection against cellular
stress is still unclear. The product(s) derived from the reactions
catalyzed by HO-1 may provide some insight. For example, the induction
of ferritin as a result of iron removal from the degradation of heme by
HO may be involved (Fig 1)
. Since the catalysis of heme by HO releases
free iron, ferritin may be induced from this released iron and serve as
a reservoir to restrict iron from reacting with both
H2O2 and superoxide resulting in decreased
production of toxic hydroxyl radicals. Furthermore, the metabolite of
heme degradation, bilirubin, is itself a potent antioxidant. Bilirubin
scavenges peroxyl radicals as efficiently as -tocopherol in
vitro, which is regarded as the most potent antioxidant of lipid
peroxidation. Bilirubin, at a concentration found in normal human
plasma, is sufficient to protect albumin from oxidation. This
antioxidant effect of bilirubin has also been demonstrated in
vivo. Another catalytic product of HO reaction that is receiving
increasing attention is carbon monoxide (CO). Although oxidation of
organic molecules contributes to the endogenous source of CO, the
predominant source of CO production, however, is from the degradation
of heme (Fig 1)
. CO, commonly classified as a toxic gas and biological
waste, is now being considered as a likely candidate molecule in the
new class of gaseous cellular messengers. Similar to nitric oxide, CO
is also capable of upregulating cyclic guanosine monophosphate via
activation of guanylate cyclase. Interestingly, nitric oxide has been
shown to also upregulate expression of HO-1 in rat aortic vascular
smooth muscle cells.10
Conclusion
Although progress has been made in our understanding of the regulation and function of HO-1 after oxidative stress,11 12 much work still lies ahead to delineate more clearly the role of HO-1 induction in oxidant-induced lung injury. The upstream signal transduction pathways regulating HO-1 gene induction remain to be more clearly defined in the various types of pulmonary cells. More importantly, a better understanding in the mechanism(s) by which HO-1 provides protection against heme and nonheme-mediated oxidant insults will potentially help us devise novel therapeutic modalities against oxidant-induced lung injury.
References
This article has been cited by other articles:
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  M. L. Jison, P. J. Munson, J. J. Barb, A. F. Suffredini, S. Talwar, C. Logun, N. Raghavachari, J. H. Beigel, J. H. Shelhamer, R. L. Danner, et al. Blood mononuclear cell gene expression profiles characterize the oxidant, hemolytic, and inflammatory stress of sickle cell disease Blood, July 1, 2004; 104(1): 270 - 280. [Abstract] [Full Text] [PDF]
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