1 Division of Nephrology, Hypertension and Transplantation, Department of Medicine, 2 Department of Neuroscience, and 3 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
First published August 9, 2001;
10.1152/ajprenal.00140.2001.Heme oxygenase-1 (HO-1) catalyzes
the rate-limiting step in heme degradation, releasing iron, carbon
monoxide, and biliverdin. Induction of HO-1 occurs as an adaptive and
protective response to several inflammatory stimuli. The transcription
factor activator protein-1 (AP-1) has been implicated in the activation
of the HO-1 gene. To elucidate the molecular mechanism of HO-1
induction, we examined the effects of diferuloylmethane (curcumin), an
inhibitor of the transcription factor AP-1. Surprisingly, curcumin by
itself was a very potent inducer of HO-1. Curcumin has
anti-inflammatory, antioxidant, and renoprotective effects. To evaluate
the mechanism of curcumin-mediated induction of HO-1, confluent human
renal proximal tubule cells were exposed to curcumin (1-8 µM).
We observed a time- and dose-dependent induction of HO-1 mRNA that was
associated with increased HO-1 protein. Coincubation of curcumin with
actinomycin D completely blocked the upregulation of HO-1 mRNA.
Blockade of nuclear factor-
B (NF-
B) with an I
B
phosphorylation inhibitor attenuated curcumin-mediated induction of
HO-1 mRNA and protein. These data demonstrate that curcumin induces
HO-1 mRNA and protein in renal proximal tubule cells. HO-1 induction by
curcumin is mediated, at least in part, via transcriptional mechanisms
and involves the NF-
B pathway.
antioxidants; nuclear factor-B; renal tubular injury
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HEME OXYGENASE-1 (HO-1) is a ~32-kDa microsomal enzyme that catalyzes the rate-limiting step in heme degradation (36). The products of the reaction are biliverdin, carbon monoxide (CO), and iron. Biliverdin is subsequently converted to bilirubin by biliverdin reductase. Two isoforms of heme oxygenase have been extensively characterized: an inducible enzyme (HO-1) and a constitutive isoform (HO-2) (4, 36). Recently, a third constitutive isoform (HO-3) has been identified (38). HO-1 is induced by several stimuli that provoke oxidant stress, including heme, hydrogen peroxide, ultraviolet (UV) radiation, heavy metals, hypoxia, hyperoxia, nitric oxide (NO), shear stress, endotoxin, cytokines, growth factors, and oxidized low-density lipoprotein (LDL) (3, 15, 21, 24, 28, 32, 59, 66). The reaction products of HO-1 have received considerable attention because they possess important antioxidant, anti-inflammatory, antiapoptotic, and possible immune modulatory effects (25, 40, 43, 48, 65). HO-1 has been implicated in several clinically relevant disorders, including acute renal failure, transplant rejection, hypertension, and atherosclerosis (27, 41, 54, 64).
Induction of HO-1 is considered to be an adaptive and beneficial response to oxidative stress in a wide variety of cells (15, 32, 47). Cytoprotective effects of HO-1 induction include degradation of the toxic heme moiety, derived from ubiquitously distributed heme proteins such as cytochromes, peroxidases, respiratory burst oxidases, pyrrolases, catalase, nitric oxide synthases (NOS), hemoglobin, and myoglobin. Furthermore, ferritin, an intracellular repository for iron, is coinduced with HO-1, allowing safe sequestering of the unbound iron liberated during the degradation of heme (11). More recently, modulation of intracellular iron stores and increased iron efflux have been suggested as a mechanism for the cytoprotective effects of HO-1 expression (22). Another favorable effect of HO-1 induction is the generation of CO, which is a signaling molecule similar to NO (19). Bilirubin has also been demonstrated to have cytoprotective properties due to its capability of scavenging peroxy radicals and inhibiting lipid peroxidation (17, 58).
The activation of HO-1 in response to most of its inducers, such as
heme, heavy metals, NO and NO donors, oxidized LDL, and cytokines,
occurs as a consequence of de novo transcription (5, 15, 21, 24,
32, 59, 66). A common denominator of stimuli that upregulate
HO-1 is a significant imposition of oxidative stress (15,
47). Consensus binding sites for nuclear factor-B (NF-
B),
activator protein-1 (AP-1), AP-2, and interleukin-6-responsive elements
as well as other transcription factors have been reported in the
promoter region of the HO-1 gene (15, 33), suggesting a
potential role for these transacting factors in modulating HO-1 gene
expression. The present investigation was designed to evaluate the
signaling pathways involved in the activation of the human HO-1 gene by
various oxidant stimuli. We therefore explored the role of curcumin, an
inhibitor of the transcription factor AP-1, in human proximal tubule
cells. Surprisingly, curcumin by itself was a potent inducer of HO-1
mRNA and protein.
Curcumin, a major constituent of the food spice turmeric, exhibits potent antioxidant, anticarcinogenic, and anti-inflammatory properties (1, 50, 53). Curcumin has a long history of medicinal use in India and Southeast Asia for a variety of inflammatory conditions and other disease pathologies (8). Curcumin inhibits lipid peroxidation and scavenges superoxide anions, hydroxyl radicals, and NO (8, 18, 53, 57). Curcumin has been shown to be cytoprotective in several models of oxidant-induced renal injury (18, 31, 55, 61). The mechanism of the cytoprotective effects of curcumin, however, is not well understood. It has been suggested that the anticarcinogenic and anti-inflammatory properties of curcumin may be due to its ability to inhibit cellular gene expression regulated by transcription factors such as Egr-1 and AP-1 (29, 45, 56).
In an attempt to elucidate the molecular mechanism and the signaling
pathway involved, we examined the effects of
N-acetylcysteine (NAC), deferoxamine (DFO), tyrosine kinase
inhibitors, actinomycin D and cycloheximide, and an inhibitor of the
NF-B pathway on curcumin-mediated HO-1 induction in human renal
proximal tubule cells.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents.
Tissue culture media, serum, and supplements were obtained from
Clonetics (Walkersville, MD). Actinomycin D, anti-human ferritin antibody, cycloheximide, curcumin, DFO, hemin, NAC, and
nordihydroguaiaretic acid (NDGA) were obtained from Sigma (St. Louis,
MO). Rabbit polyclonal antibody against rat HO-1 was from StressGen
Biotech (Vancouver, BC). BAY 11-7082, a blocker of inhibitor
IB
phosphorylation and degradation, was from Biomol Research
Laboratories, (Plymouth Meeting, PA). Tyrosine kinase inhibitors,
herbimycin and genistein were obtained from Calbiochem (San Diego, CA).
Cell culture.
Human renal proximal tubule cells (HPTC; Clonetics) were grown in renal
epithelial basal medium supplemented with fetal bovine serum (FBS;
5%), gentamicin (50 µg/ml), amphotericin B (50 µg/ml), insulin (5 µg/ml), transferrin (10 µg/ml), triiodothyronine (6.5 ng/ml),
hydrocortisone (0.5 µg/ml), epinephrine (0.5 µg/ml), and human
epidermal growth factor (10 ng/ml), at 37°C in 95% air-5% CO2. These cells have been shown to be positive for
-glutamyltranspeptidase, an enzyme marker for proximal tubule cells.
We also confirmed the presence of morphological features of proximal
tubule cells, by electron microscopy, in HPTC grown on Transwell cell
culture filters. HPTC exhibited microvilli, abundant mitochondria,
lysosomes, and endocytotic vacuoles. Studies were performed on HPTC
cultures over a range of no more than four to five passages. HK-2 cells (ATCC), an immortalized human proximal tubule epithelial cell line from
normal adult kidney (51), were grown in
keratinocyte-serum-free medium supplemented with 5 ng/ml recombinant
epidermal growth factor and 40 µg/ml bovine pituitary extract (GIBCO
BRL, Rockville, MD). Cells were grown in 100-mm tissue culture plates
and studied as a confluent monolayer in all experiments. Twenty-four
hours before stimulation, media were changed in HPTC to complete media containing 0.5% FBS.
Northern analysis.
Total cellular RNA was extracted from cultured cells using the method
of Chomczynski and Sacchi (16). The RNA was
electrophoresed on 1% agarose-formaldehyde gels, blotted onto nylon
membranes, and hybridized to a 32P-labeled cDNA probe for
human HO-1. To control for loading and transfer of RNA, the blots were
reprobed with a cDNA probe for human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Autoradiography was performed with the use of
intensifying screens at 80°C. Densitometry was performed using
National Institutes of Health Image 1.60 software on a Power Macintosh computer.
Immunoblot analysis. For ferritin immunoblots, cells were washed and collected in a solution of 8 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, and 140 mM NaCl, and centrifuged at 1,000 g for 2 min. The pellet was resuspended in potassium phosphate buffer, pH 7.4, containing 10 mM EDTA, sonicated for 20 s, and centrifuged at 10,000 g, and then the supernatant was collected for Western blot analysis. For HO-1 immunoblots, cells were washed twice with ice-cold PBS and lysed in Triton lysis buffer. Protein concentrations were determined by bicinchoninic acid assay (Pierce, Rockford, IL). Samples (20 µg) were separated in a 10 (for HO-1) or 15% (for ferritin) SDS-polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane. The membranes were incubated for 1.5 h with a rabbit polyclonal antibody against rat HO-1 (1:500 dilution) or anti-human ferritin antibody (1:2,000) followed by incubation with peroxidase-conjugated goat anti-rabbit IgG antibody (1:20,000 dilution) for 1 h. Labeled protein bands were examined using chemiluminescence (Pierce).
Data analysis. Data are expressed as means ± SE. Statistical analysis of HO-1 mRNA levels in the different groups was performed using ANOVA and the Student-Newman-Keuls test. All results were considered significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Curcumin induces HO-1 mRNA and protein expression in human renal
proximal tubule cells.
Curcumin has previously been shown to inhibit the transcription
factor AP-1 (45, 56). AP-1 has been implicated in the activation of the HO-1 gene (7, 15). Therefore, we
determined the effects of curcumin on stimulus-mediated HO-1 induction
in HPTC. Interestingly, curcumin alone induced HO-1 mRNA and protein in
HPTC. A 4-h exposure of HPTC to curcumin at concentrations of 1.0, 2.0, 4.0, and 8.0 µM resulted in a dose-dependent increase (up to 12-fold)
of HO-1 mRNA (Fig. 1A).
Immunoblot analysis demonstrated that induction of HO-1 mRNA by
curcumin (8.0 µM) was accompanied by a fivefold increase in HO-1
protein (Fig. 1B). Induction of HO-1 mRNA was observed as
early as 2 h, and peaked at 6 h, in the presence of curcumin
(8.0 µM) (Fig. 1C). Ethanol (0.01%, vehicle) alone did
not induce HO-1 mRNA. Curcumin (8.0 µM, 4 h) also resulted in a
significant induction of HO-1 mRNA in HK-2 cells, an immortalized human
proximal tubule cell line (Fig. 1D). No cytotoxicity was observed by using the above concentrations of curcumin for up to
24 h.
|
Induction of HO-1 mRNA by curcumin requires RNA synthesis.
To evaluate the mechanism of curcumin-mediated HO-1 induction,
confluent HPTC were cotreated with 8 µM curcumin and 4 µM
actinomycin D, a transcriptional inhibitor, or 20 µM cycloheximide,
an inhibitor of protein synthesis, for 4 h. Actinomycin D
completely blocked curcumin-mediated HO-1 mRNA induction (Fig.
2). Cycloheximide had no significant
effect on the upregulation of HO-1 mRNA steady-state levels in response
to curcumin (Fig. 2). These data suggest that HO-1 induction by
curcumin is dependent on RNA synthesis and is not dependent on de novo
protein synthesis.
|
Curcumin-induced HO-1 gene expression is not dependent on increased
mRNA stability.
To examine whether increased HO-1 gene expression was a result of
increased mRNA stability, confluent HPTC were preincubated with
curcumin followed by the addition of actinomycin D. Cells were
incubated with 8 µM curcumin for 4 h, washed, and exposed to
media containing 4 µM actinomycin D in the absence or presence of an
additional dose of 8 µM curcumin. RNA was isolated and processed at
0.5, 1, 2, 4, 8, 16, and 24 h, respectively. No significant change
in the half-life of HO-1 mRNA was observed in the actinomycin D-treated
cells in the presence or absence of the second dose of curcumin (Fig.
3). These data indicate that the
half-life of curcumin-mediated HO-1 mRNA induction is ~1.8 h and is
not dependent on increases in mRNA stability.
|
Curcumin induces ferritin protein expression in HPTC.
Previous studies have shown that, in several instances, induction of
HO-1 is coupled to the induction of ferritin, which safely sequesters
the iron released during heme degradation (3, 11, 41).
Therefore, we examined the effects of 8 µM curcumin on ferritin
induction. Immunoblot analysis demonstrated a 2.6-fold induction of
light-chain ferritin after a 72-h exposure to curcumin in HPTC (Fig.
4). A modest increase in heavy chain
ferritin was also observed, represented by the band at ~21 kDa (Fig.
4).
|
Antioxidants and tyrosine kinase inhibitors do not inhibit
curcumin-mediated induction of HO-1 mRNA.
NAC, an antioxidant, has been reported to block induction of HO-1 by
the cytokines tumor necrosis factor (TNF)- and interleukin (IL)-1
in endothelial cells (59). In addition, we have previously reported that DFO, an iron chelator, inhibited HO-1 induction by
oxidized LDL and hyperoxia in endothelial cells (3, 23). We examined the effects of the above agents on curcumin-mediated induction of HO-1 mRNA. HPTC were pretreated with 500 µM DFO
overnight or 50 µM and 1 mM NAC for 1 h. Cotreatment of DFO or
NAC with 8 µM curcumin for 4 h had no effect on
curcumin-mediated HO-1 mRNA induction (Fig.
5A). Therefore, induction of
HO-1 by curcumin appears to be through a mechanism independent of overt
oxidative stress and the generation of reactive oxygen species.
|
The NF-B pathway is involved in curcumin-mediated induction of
HO-1 mRNA and protein.
At high concentrations (20-60 µM), curcumin is an inhibitor of
NF-
B, and at lower concentrations (<10 µM) it selectively inhibits AP-1 (6, 35, 45). NF-
B is normally present in the cytosol, where it is bound to an inhibitory protein component, I
B (35). I
B undergoes phosphorylation, ubiquination,
and degradation when activated, thereby releasing active NF-
B, which
then translocates into the nucleus (9, 20). Coincubation
of HPTC with curcumin (8 µM) and BAY 11-7082 (10 µM), an inhibitor
of I
B phosphorylation (46), abolished curcumin-mediated
induction of HO-1 mRNA (Fig. 6,
A and B) and protein (Fig. 6C). BAY
11-7082 (10 µM) alone had no effect on HO-1 induction.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There is considerable evidence to support the involvement of reactive oxygen species in the pathogenesis of renal tubular injury from ischemia-reperfusion and nephrotoxins (10, 44, 52, 63). While measures of free radical generation and lipid peroxidation are increased, a variety of antioxidants and several oxidant scavenging enzymes have been shown to attenuate the course of renal injury (10, 63). In such states of increased oxidative stress, cells and tissues gear up inherent defense systems as an adaptive response. One such antioxidant response in renal tubules exposed to injurious stimuli is the robust and dramatic induction of HO-1. Induction of HO-1 occurs in several models of acute renal injury and plays a cytoprotective role (41, 42, 54). Studies using chemical inducers or inhibitors as well as studies in HO-1-knockout mice provide evidence for a protective role of HO-1 in renal injury (42, 54). The induction of HO-1 has been recognized as a characteristic cellular response to a diverse array of potentially injurious stimuli in several nonrenal biological systems as well (15, 43, 65). Besides its heme- degrading function, several of the by-products of the HO-1 catalyzed reaction have been shown to have antioxidant, anti-inflammatory, as well as antiapoptotic effects (25, 40, 43, 48, 65).
The molecular mechanisms underlying HO-1 induction are complex and
tightly regulated at the level of transcription. In the case of the
mouse HO-1 gene, Alam and colleagues (7, 15) have
implicated AP-1 and related transcription factors in the activation of
the HO-1 gene after oxidant stressors. In an attempt to evaluate the
role of AP-1 in the induction of the HO-1 gene in human proximal tubule
cells, we were surprised to find that low doses of curcumin, an AP-1
inhibitor, upregulated HO-1 mRNA and protein. Motterlini et al.
(39) have recently reported the induction of HO-1 by
curcumin in bovine endothelial cells. Furthermore, they have observed
that prior induction of HO-1 by curcumin rendered significant
resistance of cells to glucose oxidase-induced cell damage after a
hypoxic stimulus (39). The observation that an inhibitor
(curcumin) of a transcription factor (AP-1) induces expression of HO-1
is similar to the findings of Hartsfield et al. (26). They
have reported dramatic induction of HO-1 with pyrrolidine
dithiocarbamate (PDTC), an inhibitor of NF-B, while they were
studying the involvement of the NF-
B pathway in NO-mediated induction of HO-1. In addition, these authors have demonstrated that
PDTC-mediated induction of HO-1 occurred via AP-1 (26).
Curcumin, a polyphenolic compound, belongs to the bioflavanoid family and exhibits potent antioxidant, anticarcinogenic, and anti-inflammatory properties (1, 8, 18, 50, 53, 57). The beneficial effects of curcumin have been well documented in both renal and nonrenal models of oxidant injury (18, 31, 55, 60, 61). However, the exact mechanism of the beneficial effects is not clearly known. Curcumin inhibits lipid peroxidation and scavenges superoxide anions, hydroxyl radicals, and NO (18, 53, 57). Curcumin and a related bioflavanoid, quercetin, have also been shown to decrease immune- and nonimmune-mediated renal injury in ischemia reperfusion (55). When used either alone or in combination with mycophenolate mofetil, quercetin and curcumin reduce renal injury and attenuate expression of proinflammatory cytokines (55). More recently, curcumin has been shown to induce the expression of HO-1 in both normal and obstructed rat kidneys (31), providing in vivo application to the use of curcumin as a relevant HO-1 inducer. The localization of such induction in the kidney has not been reported yet and would be of interest. Our studies have also demonstrated the induction of ferritin by curcumin. Although we have not evaluated the mechanism of this induction, in most instances ferritin is coinduced with HO-1 (11, 41, 62). However, inhibition of HO-1 activity does not always result in decreased ferritin content (2, 12), suggesting that increased ferritin can occur via mechanisms independent of iron release from the HO-1 reaction. Given that curcumin induces HO-1, it is tempting to speculate that the antioxidant and anti-inflammatory properties exhibited by curcumin may be mediated, at least in part, through HO-1 induction.
At the cellular level, curcumin has been reported to inhibit expression
of genes such as inducible NOS, VCAM-1, E-selectin, and others via
suppression of the AP-1 transcription factor (6, 13, 14,
29). AP-1 proteins are regulated by several growth factors,
cytokines, and by various agents that induce oxidative stress. Curcumin
at low concentrations (1-10 µM) is an AP-1 inhibitor; however,
at higher doses, it also blocks several reactions in which NF-B
plays a major role (6). Pendurthi et al.
(45), using a human umbilical vein endothelial cell line,
have demonstrated that curcumin inhibits phorbol 12-myristate
13-acetate (PMA)-, lipopolysaccharide-, TNF-
-, and thrombin-induced
tissue factor gene expression. Curcumin (1-30 µM) suppresses the
constitutive binding of c-Jun/c-Fos to the AP-1 site of tissue factor
and completely inhibits PMA-mediated induction of tissue factor. Higher
concentrations of curcumin (30-40 µM) block the activation of
NF-
B by the various inducers via suppression of I
B
phosphorylation (45). Similarly, Ahmad et al.
(6), using human microvascular endothelial cells, have
shown that low concentrations of curcumin (10 µM) inhibit TNF-
-induced activation of AP-1 but do not affect
TNF-
-induced activation of NF-
B. Similar results have
been obtained with NDGA, another AP-1 inhibitor (6). In
experiments using NDGA, we have observed that this compound also
induces HO-1 mRNA in HPTC.
Our studies suggest that the NF-B pathway is involved in curcumin-
and NDGA-mediated induction of HO-1 in human renal epithelial cells.
NF-
B proteins reside in the cytoplasm and, when activated, are
translocated to the nucleus. Translocation is induced by several proinflammatory stimuli, including cytokines (TNF, IL-1), bacterial products, protein synthesis inhibitors, oxidative stress, UV light, and
phorbol esters. NF-
B activation controls the induction of several
genes involved in the inflammatory, immune, and acute phase response,
as well as inhibition of apoptosis (9). Most of
the studies that report the inhibitory effects of curcumin on NF-
B
activation have been performed on cancer cell lines (56). Whereas high concentrations of curcumin (40-60 µM) inhibit TNF-, hydrogen peroxide-, and phorbol ester-mediated induction of NF-
B, lower concentrations of curcumin (2-20 µM) actually show a mild increase in NF-
B activation (56). NF-
B is present in
the cytoplasm of unstimulated cells as a p50/p65 heterodimer complexed
with I
B
, the inhibitory subunit that prevents migration of
NF-
B to the nucleus. Translocation of free NF-
B to the nucleus is preceded by the phosphorylation and degradation or dissociation of
I
B
(30). A wide array of stimuli activate I
B
kinase, which phosphorylates I
B (20). I
B
is
phosphorylated rapidly (within 5 min) after TNF treatment
(46). The three inducers of NF-
B that were used, TNF,
phorbol ester, and hydrogen peroxide, are known generators of reactive
oxygen species, and it is possible that the effects of higher
concentrations of curcumin on inhibition of NF-
B activation are
mediated via quenching of these reactive oxygen intermediates. Pierce
et al. (46) have identified two novel inhibitors of
I
B
phosphorylation, which inhibited cytokine-induced expression
of adhesion molecules such as intercellular adhesion molecule-1,
VCAM-1, and E-selectin. Significant anti-inflammatory effects have also
been observed in vivo with a related compound (46).
Reversible activation of stress-activated kinases p38 and JNK-1 and
activation of tyrosine kinase have been shown with these compounds
(46). The authors suggest that these compounds do not act
as global inhibitors of cytokine-induced phosphorylation events but
selectively inhibit phosphorylation of I
B
(46). In
the present studies, inhibition of NF-
B, using one of the novel
inhibitors of I
B
phosphorylation, BAY 11-7082 (10 µM), completely blocked curcumin-mediated HO-1 mRNA and protein induction. It is interesting that curcumin, on the one hand, induces HO-1 via a
proinflammatory transcription factor (NF-
B) but, on the other hand,
exhibits antioxidant and anti-inflammatory properties, further
emphasizing the functional significance of HO-1 as an adaptive response
to inflammation.
In an effort to identify regulatory sequences that control
curcumin-mediated HO-1 induction in HPTC, we have evaluated up to 4.5
kb of the human HO-1 promoter region (containing consensus binding
sites for AP-1 and NF-
B). This fragment responds partially to known
inducers of the gene (e.g. heme and cadmium) but lacks the
cis-acting elements necessary for curcumin-mediated
induction of HO-1 (data not shown). The absence of a response with the
4.5-kb human HO-1 promoter region to curcumin is further corroborated by the failure of this fragment to respond to other stimuli that directly increase de novo HO-1 gene transcription, i.e., oxidized lipids, hydrogen peroxide, hyperoxia, and iron/hyperoxia (5, 23). Based on the results of our present study, which implicates NF-
B as a potential transcription factor involved in
curcumin-mediated HO-1 induction, it is possible that an important
NF-
B site(s) outside of the
4.5-kb promoter region may be
responsible for HO-1 gene induction. Further studies using chromatin
structure analysis to delineate the regions around the human HO-1 gene
that control curcumin-mediated HO-1 induction would be of interest.
The tyrosine kinase pathway has also been implicated in the induction of HO-1 by hemin, cadmium, and sodium arsenite (37). Bioflavanoids, such as quercetin and curcumin, have a common polyphenolic structure and, in addition to inhibiting transcription factors, are also broadly acting inhibitors of both cytosolic and membrane tyrosine kinases. Curcumin has been shown to inhibit the activities of cellular kinases, including protein kinase C and tyrosine protein kinase, and to be a potent inhibitor of the initiation, promotion, and progression of several types of human cancers (49). In the present studies, the tyrosine kinase inhibitors herbimycin and genistein had no effect on curcumin-mediated HO-1 induction, suggesting that this pathway is not involved.
Changes in cellular oxidative stress are critical in the induction of
the HO-1 gene by several stimuli. However, similar to the observations
of Motterlini et al. (39), we have not observed any
inhibition of curcumin-mediated HO-1 induction by the antioxidant NAC.
These results are similar to the mechanism of HO-1 gene induction by
transforming growth factor- that seems to be independent of changes
in the cellular redox state (28). In addition, unlike the
effects of DFO on oxidized LDL (3)- or hyperoxia
(23)-mediated induction of HO-1, we have not observed any
effect of DFO on curcumin-mediated HO-1 induction. These data suggest
that the induction of HO-1 may not always represent an index of changes
in the cellular redox status and that other nonredox-related pathways
may exist and need further elucidation.
In summary, curcumin induces HO-1 mRNA and protein as well as ferritin
protein in HPTCs. HO-1 induction by curcumin is mediated, at least in
part, via transcriptional mechanisms and most likely involves the
NF-B pathway. The beneficial effects of curcumin may, in part, be
mediated through an increase in HO-1 gene expression, and further
studies to explore this correlation are necessary in models of renal injury.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants K08 DK-02446 and R03 DK-56279 (to A. Agarwal). N. Hill-Kapturczak was supported by a postdoctoral training grant (T32 DK-07518) to the Division of Nephrology, University of Florida. We are grateful to Leigh Truong and Supriya Prabhu for helpful contributions to this work.
![]() |
FOOTNOTES |
---|
First published August 9, 2001; 10.1152/ajprenal.00140.2001
Address for reprint requests and other correspondence: A. Agarwal, Assistant Professor of Medicine, Division of Nephrology, Hypertension and Transplantation, Box 100224 JHMHC, 1600 SW Archer Road, University of Florida, Gainesville FL 32610 (Email: agarwal{at}nersp.nerdc.ufl.edu).
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.
Received 7 May 2001; accepted in final form 5 July 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abe, Y,
Hashimoto S,
and
Horie T.
Curcumin inhibition of inflammatory cytokine production by human peripheral blood monocytes and alveolar macrophages.
Pharmacol Res
39:
41-47,
1999[ISI][Medline].
2.
Agarwal, A,
Balla J,
Alam J,
Croatt AJ,
and
Nath KA.
Induction of heme oxygenase in toxic renal injury: a protective role in cisplatin nephrotoxicity in the rat.
Kidney Int
48:
1298-1307,
1995[ISI][Medline].
3.
Agarwal, A,
Balla J,
Balla G,
Croatt AJ,
Vercellotti GM,
and
Nath KA.
Renal tubular epithelial cells mimic endothelial cells upon exposure to oxidized LDL.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F814-F823,
1996
4.
Agarwal, A,
and
Nick HS.
Renal response to tissue injury: lessons from heme oxygenase-1 gene ablation and overexpression.
J Am Soc Nephrol
11:
965-973,
2000
5.
Agarwal, A,
Shiraishi F,
Visner GA,
and
Nick HS.
Linoleyl hydroperoxide transcriptionally upregulates heme oxygenase-1 gene expression in human renal epithelial and aortic endothelial cells.
J Am Soc Nephrol
9:
1990-1997,
1998[Abstract].
6.
Ahmad M, Theofanidis P, and Medford RM. Role of activating
protein-1 in the regulation of the vascular cell adhesion molecule-1
gene expression by tumor necrosis factor-. J Biol
Chem 4616-4621, 1998.
7.
Alam, J,
Camhi S,
and
Choi AMK
Identification of a second 5' distal region that functions as a basal and inducer-dependent transcriptional enhancer of the mouse heme oxygenase-1 gene.
J Biol Chem
270:
11977-11984,
1995
8.
Ammon, HPT,
and
Wahl MA.
Pharmacology of Curcuma longa.
Planta Med
57:
1-7,
1991[ISI][Medline].
9.
Baeuerle, PA,
and
Baltimore D.
IB: a specific inhibitor of the NF-
B transcription factor.
Science
242:
540-546,
1988[ISI][Medline].
10.
Baliga, R,
Ueda N,
Walker PD,
and
Shah SV.
Oxidant mechanisms in toxic acute renal failure.
Drug Metab Rev
31:
971-997,
1999[ISI][Medline].
11.
Balla, G,
Jacob HS,
Balla J,
Rosenberg M,
Nath KA,
Apple F,
Eaton JW,
and
Vercellotti GM.
Ferritin: a cytoprotective antioxidant strategem of endothelium.
J Biol Chem
267:
18148-18153,
1992
12.
Balla, J,
Nath KA,
Balla G,
Juckett MB,
Jacob HS,
and
Vercellotti GM.
Endothelial cell heme oxygenase and ferritin induction in rat lung by hemoglobin in vivo.
Am J Physiol Lung Cell Mol Physiol
268:
L321-L327,
1995
13.
Brouet, I,
and
Ohshima H.
Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages.
Biochem Biophys Res Commun
206:
533-540,
1995[ISI][Medline].
14.
Chan, MM,
Huang HI,
Fenton MR,
and
Fong D.
In vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with anti-inflammatory properties.
Biochem Pharmacol
55:
1955-1962,
1998[ISI][Medline].
15.
Choi, AMK,
and
Alam J.
Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury.
Am J Respir Cell Mol Biol
15:
9-19,
1996[Abstract].
16.
Chomczynski, P,
and
Sacchi N.
Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
17.
Clark, JE,
Foresti R,
Sarathchandra P,
Kaur H,
Green CJ,
and
Motterlini R.
Heme oxygenase-1 derived bilirubin ameliorates post-ischemic myocardial dysfunction.
Am J Physiol Heart Circ Physiol
278:
H643-H651,
2000
18.
Cohly, HHP,
Taylor A,
Angel MF,
and
Salahudeen AK.
Effect of turmeric, turmerin and curcumin on H2O2-induced renal epithelial (LLC-PK1) cell injury.
Free Radic Biol Med
24:
49-54,
1998[ISI][Medline].
19.
Dawson, TM,
and
Snyder SH.
Gases as biological messengers: nitric oxide and carbon monoxide in the brain.
J Neurol
14:
5147-5159,
1994.
20.
DiDonato, JA,
Hayakawa M,
Rothwarf DM,
Zandi E,
and
Karrin M.
A cytokine-responsive IB kinase that activates the transcription factor NF-
B.
Nature
388:
548-554,
1997[ISI][Medline].
21.
Durante, W,
Kroll MH,
Christodoulides N,
Peyton KJ,
and
Schafer AI.
Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells.
Circ Res
80:
557-564,
1997
22.
Ferris, CD,
Jaffrey SR,
Sawa A,
Takahashi M,
Brady SD,
Barrow RK,
Tysoe SA,
Wolosker H,
Baranano DE,
Dore S,
Poss KD,
and
Snyder SH.
Haem oxygenase-1 prevents cell death by regulating cellular iron.
Nature
1:
152-157,
1999.
23.
Fogg, S,
Agarwal A,
Nick HS,
and
Visner GA.
Iron regulates hyperoxia-dependent human heme oxygenase 1 gene expression in pulmonary endothelial cells.
Am J Respir Cell Mol Biol
20:
797-804,
1999
24.
Foresti, R,
Clark JE,
Green CJ,
and
Motterlini R.
Thiol compounds interact with nitric oxide in regulating heme oxygenase-1 induction in endothelial cells. Involvement of superoxide and peroxynitrite anions.
J Biol Chem
272:
18411-18417,
1997
25.
Hancock, WW,
Buelow R,
Sayegh MH,
and
Turka LA.
Antibody-induced transplant arteriosclerosis is prevented by graft expression of antioxidant and anti-apoptotic genes.
Nat Med
4:
1392-1396,
1998[ISI][Medline].
26.
Hartsfield, CL,
Alam J,
and
Choi AM.
Transcriptional regulation of the heme oxygenase 1 gene by pyrrolidine dithiocarbamate.
FASEB J
12:
1675-82,
1998
27.
Haugen, EN,
Croatt AJ,
and
Nath KA.
Angiotensin II induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro.
Kidney Int
58:
144-152,
2000[ISI][Medline].
28.
Hill-Kapturczak, N,
Truong L,
Thamilselvan V,
Visner GA,
Nick HS,
and
Agarwal A.
Smad7-dependent regulation of heme oxygenase-1 by transforming growth factor- in human renal epithelial cells.
J Biol Chem
275:
40904-40909,
2000
29.
Huang, RS,
Lee SC,
and
Lin JK.
Suppression of c-jun/AP-1 activation by an inhibitor of tumor promotion in mouse fibroblast cells.
Proc Natl Acad Sci USA
88:
5292-5296,
1991[Abstract].
30.
Imbert, V,
Rupec RAL,
Livolsi A,
Pahl HL,
Traencker EBM,
Mueller-Dieckmann C,
Farhifar DRB,
Auberger P,
Baeuerle PA,
and
Peyron JF.
Tyrosine phosphorylation of IB-
activates NF-
B without proteolytic degradation of I
B-
.
Cell
86:
787-798,
1996[ISI][Medline].
31.
Jones, EA,
Shahed A,
and
Shoskes DA.
Modulation of apoptotic and inflammatory genes by bioflavonoids and angiotensin II inhibition in ureteral obstruction.
Urology
56:
346-351,
2000[ISI][Medline].
32.
Keyse, SM,
and
Tyrrell RM.
Heme oxygenase is the major 32-kDa stress protein induced in skin fibroblasts by UVA radiation, hydrogen peroxide and sodium arsenite.
Proc Natl Acad Sci USA
86:
99-103,
1989[Abstract].
33.
Lavrovsky, Y,
Schwartzman ML,
Levere RD,
Kappas A,
and
Abraham NG.
Identification of binding sites for transcription factors NF-kappa B and AP-2 in the promoter region of the human heme oxygenase-1 gene.
Proc Natl Acad Sci USA
91:
5987-5991,
1994[Abstract].
34.
Liou, HC,
and
Baltimore D.
Regulation of the NF-B/rel transcription factor and I
B inhibitor system.
Curr Opin Cell Biol
5:
477-487,
1993[Medline].
35.
Luo, Y,
Hattori A,
Munoz J,
Qin ZH,
and
Roth GS.
Intrastriatal dopamine injection induces apoptosis through oxidation-involved activation of transcription factors AP-1 and NF-B in rats.
Mol Pharmacol
56:
254-264,
1999
36.
Maines, MD.
The heme oxygenase system: a regulator of second messenger gases.
Annu Rev Pharmacol Toxicol
37:
517-554,
1997[ISI][Medline].
37.
Masuya, Y,
Hioki K,
Tokunaga R,
and
Taketani S.
Involvement of the tyrosine phosphorylation pathway in induction of human heme oxygenase-1 by hemin, sodium arsenite, and cadmium chloride.
J Biochem (Tokyo)
124:
628-633,
1998[Abstract].
38.
McCoubrey, WK,
Huang TJ,
and
Maines MD.
Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3.
Eur J Biochem
247:
725-732,
1997[Abstract].
39.
Motterlini, R,
Foresti R,
Bassi R,
and
Green CJ.
Curcumin, an antioxidant and anti-inflammatory agent induces heme oxygenase-1 and protects endothelial cells against oxidative stress.
Free Radic Biol Med
28:
1303-1312,
2000[ISI][Medline].
40.
Nath, KA.
Heme oxygenase-1: a redoubtable response that limits reperfusion injury in the transplanted adipose liver.
J Clin Invest
104:
1485-1486,
1999
41.
Nath, KA,
Balla G,
Vercellotti GM,
Balla J,
Jacob HS,
Levitt MD,
and
Rosenberg ME.
Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat.
J Clin Invest
90:
267-270,
1992[ISI][Medline].
42.
Nath, KA,
Haggard JJ,
Croatt AJ,
Grande JP,
Poss KD,
and
Alam J.
The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo.
Am J Pathol
156:
1527-1535,
2000
43.
Otterbein, LE,
Mantell LL,
and
Choi AMK
Carbon monoxide provides protection against hyperoxic lung injury.
Am J Physiol Lung Cell Mol Physiol
276:
L688-L694,
1999
44.
Paller, MS.
The cell biology of reperfusion injury in the kidney.
J Investig Med
42:
632-639,
1994[ISI][Medline].
45.
Pendurthi, UR,
Williams JT,
and
Rao LVM
Inhibition of tissue factor gene activation in cultured endothelial cells by curcumin: suppression of activation of transcription factors Egr-1, AP-1, and NF-B.
Arterioscler Thromb Vasc Biol
17:
3406-3413,
1997
46.
Pierce, JW,
Schoenleber R,
Jesmok G,
Best J,
Moore SA,
Collins T,
and
Gerritsen ME.
Novel inhibitors of cytokine-induced IB
phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo.
J Biol Chem
272:
21096-21103,
1997
47.
Platt, JL,
and
Nath KA.
Heme oxygenase: protective gene or Trojan horse.
Nat Med
4:
1364-1365,
1998[ISI][Medline].
48.
Poss, KD,
and
Tonegawa S.
Reduced stress defense in heme oxygenase-1 deficient cells.
Proc Natl Acad Sci USA
94:
10925-10930,
1997
49.
Rao, CV,
Rivenson A,
Simi B,
and
Reddy BS.
Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound.
Cancer Res
55:
259-266,
1995[Abstract].
50.
Ruby, AJ,
Kuttan G,
Babu KD,
Rajasekharan KN,
and
Kuttan R.
Anti-tumour and antioxidant activity of natural curcuminoids.
Cancer Lett
94:
79-83,
1995[ISI][Medline].
51.
Ryan, MJ,
Johnson G,
Kirk J,
Fuerstenberg SM,
Zager RA,
and
Torok-Storb B.
HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney.
Kidney Int
45:
48-57,
1994[ISI][Medline].
52.
Salahudeen, AK,
Clark EC,
and
Nath KA.
Hydrogen peroxide-induced renal injury. A protective role for pyruvate in vitro and in vivo.
J Clin Invest
88:
1886-1893,
1991[ISI][Medline].
53.
Shalini, VD,
and
Srinivas L.
Lipid peroxide induced DNA damage: protection by turmeric (Curcuma longa).
Mol Cell Biochem
77:
3-10,
1987[ISI][Medline].
54.
Shiraishi, F,
Curtis LM,
Truong L,
Poss K,
Visner GA,
Madsen K,
Nick HS,
and
Agarwal A.
Heme oxygenase-1 gene ablation or expression modulates cisplatin-induced renal tubular apoptosis.
Am J Physiol Renal Physiol
278:
F726-F736,
2000
55.
Shoskes, DA.
Effect of bioflavanoids quercetin and curcumin on ischemic renal injury: a new class of renoprotective agents.
Transplantation
66:
147-152,
1998[ISI][Medline].
56.
Singh, S,
and
Aggarwal BB.
Activation of transcription factor NF-B is supported by curcumin (diferuloylmethane).
J Biol Chem
270:
24995-25000,
1995
57.
Sreejayan Rao, MN.
Nitric oxide scavenging by curcuminoids.
J Pharm Pharmacol
49:
105-107,
1997[ISI][Medline].
58.
Stocker, R,
Yamamoto Y,
McDonagh AF,
Glazer AN,
and
Ames BN.
Bilirubin is an antioxidant of possible physiologic significance.
Science
235:
1043-1046,
1987[ISI][Medline].
59.
Terry, CM,
Clikeman JA,
Hoidal JR,
and
Callahan KS.
TNF- and IL-1
induce heme oxygenase-1 via protein kinase C, Ca2+, and phospholipase A2 in endothelial cells.
Am J Physiol Heart Circ Physiol
276:
H1493-H1501,
1999
60.
Venkatesan, N.
Curcumin attenuation of acute adriamycin myocardial toxicity in rats.
Br J Pharmacol
124:
425-427,
1998[Abstract].
61.
Venkatesan, N,
Punithavathi D,
and
Arumugam V.
Curcumin prevents adriamycin nephrotoxicity in rats.
Br J Pharmacol
129:
231-234,
2000
62.
Vile, GF,
and
Tyrrell RM.
Oxidative stress resulting from ultraviolet A irradiation of human skin fibroblasts leads to a heme oxygenase-dependent increase in ferritin.
J Biol Chem
268:
14678-14681,
1993
63.
Walker, PD,
and
Shah SV.
Gentamicin-enhanced production of hydrogen peroxide by renal cortical mitochondria.
Am J Physiol Cell Physiol
253:
C495-C499,
1987
64.
Wang, LJ,
Lee TS,
Lee FY,
Pai RC,
and
Chau LY.
Expression of heme oxygenase-1 in atherosclerotic lesions.
Am J Pathol
152:
711-720,
1998[Abstract].
65.
Willis, D,
Moore AR,
Frederick R,
and
Willoughby DA.
Heme oxygenase: a novel target for the modulation of the inflammatory response.
Nat Med
2:
87-90,
1996[ISI][Medline].
66.
Yet, SF,
Pellacani A,
Patterson C,
Tan L,
Folta SC,
Foster L,
Lee WS,
Hsieh CM,
and
Perrella MA.
Induction of heme oxygenase-1 expression in vascular smooth muscle cells. A link to endotoxic shock.
J Biol Chem
272:
4295-4301,
1997