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Nuclear factor kappa B (NF-kB) is a collective name for inducible dimeric transcription factors comprising of members of
the Rel family. Eight members of the Rel protein family including
c-Rel, NF-kB1 (p50/p105), NF-kB2 (p52/p100), Rel A (p65), Rel
B, the Drosophila proteins Dorsal, Dif, and Relish have been cloned and
characterized[1], and all share a conserved Rel
homology domain (RHD) that is responsible for dimerization, nuclear localization, and DNA binding/transcription activation.
NF-kB is found in essentially all cell types and it helps to govern the expression of genes involved in such disparate
processes as growth, development, inflammatory/immune response, auto-regula-tion, and transcription of viral
genomes[2]. Some studies have also shown that
NF-kB plays an important role in the expression of matrix metalloproteinases
(MMP)[3,4]. A family of structurally related proteins known as the inhibitors of
NF-kB (IkB) are responsible for the regulation of the DNA
binding activity and nucleo/cytoplasmic distribution of
NF-kB. All IkB share an ankyrin repeat domain (ARD) that interfaces
with the RHD of NF-kB homo/heterodimers, forming a stable inhibited complex.
IkBa binds the p65/p50 heterodimer (the most ubiquitous and biologically active
NF-kB dimmer) and p65/c-Rel heterodimers, and is the best characterized protein in
this family. The association IkBa with NF-kB disrupts DNA binding and masks the nuclear localization signals (NLSs)
located in the C-terminal region of the RHD, which is believed to impede nuclear translocation of
NF-kB[5]. So NF-kB is normally sequestered in the cytoplasm of non-stimulated cells and consequently must be translocated into the nucleus to
function. A variety of extracellular stimuli including in tumor necrosis factor
(TNF)-a and interleukin (IL)-1 cause the rapid phosphorylation, ubiquitination, and ultimately proteolytic degradation of
IkBa, which allows NF-kB to translocate to the nucleus where it regulates gene
transcription[6,7]. The multisubunit
IkB kinase (IKK) responsible for inducible
IkBa phosphorylation is the point of convergence for most
NF-kB-activating stimuli. The IKK complex consists of 2 catalytic subunits,
IKKa and IKKb, and a scaffold molecule, the NF-kB essential modulator (or
IKKb). Gene depletion studies have demonstrated that
IKKb, but not IKKa, plays an essential role in
NF-kB activation mediated by LPS, TNF-a, and
IL-1[8_10].
MMP that can degrade most components of the extracellular matrix (ECM) play an important role in the pathogenesis of
chronic obstructive pulmonary disease
(COPD)[11,12]. Ya-qing LI, et al
(unpublished data), have shown that
N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) can down-regulate the expression of MMP-9 induced by
TNF-a in alveolar macrophages (AM) from patients with COPD via a
NF-kB-mediated signal pathway. Because NAC and PDTC are
antioxidants, and both can inhibit the activation of
NF-kB, it has been postulated that reactive oxygen species (ROS) may act
as second messengers leading to NF-kB
activation[13]. However, this hypothesis does not explain why other antioxidants,
such as epigallocatechin-gallate (EGCG) and trolox, failed to inhibit
NF-kB activation induced by TNF-a[14]. Therefore it was
necessary to study the pharmacologicalmechanisms of NAC and PDTC as inhibitors of
NF-kB-mediated pathways. So in the present study, their effects on the phosphorylation of
IKKb, IKKa, and IkBa were investigated, and NF-kB activity induced
by TNF-a and IL-1 was also explored.
Materials and methods
Experimental subjects Twenty patients with COPD were recruited from Tongji Hospital, Wuhan, China. The diagnosis of
COPD was made on the basis of medical history, clinical symptoms and spirometric data, according to the criteria of the Global
Initiative for Chronic Obstructive Lung
Disease[15]. Pulmonary function tests were performed to determine forced vital
capacity (FVC) and forced expiratory volume in one second
(FEV1). Chronic airflow obstruction was defined as having an
FEV1/FVC ratio of <70% and a postbronchodilator
FEV1 value that was <80% of the predicted value. Subjects were excluded
if they had asthma, recent exacerbation of COPD, or had received recent treatment with oral corticosteroids. All patients gave
written informed consent before participating in the study, which was approved by the Ethics Committee of Tongji Hospital.
Bronchoalveolar lavage and AM culture
Bronchoscopy and bronchoalveolar lavage
(BAL)were performed as described
previously[16]. Three 30 mL aliquots of sterile saline solution were instilled using a bronchoscope to the subseg-mental
bronchi of the middle lobe. The recovered fluid was strained through surgical gauze to remove debris and mucus, and was
then centrifuged (400×g for 10 min). BAL cells were washed twice in phosphate-buffered saline (PBS), and resuspended in
RPMI-1640 medium (Hyclone, Logan, Utah, USA) containing 10% fetal calf serum (FCS, Gibco, Grand Island, New York,
USA). The cells were allowed to adhere for 2.5 h at 37 °C in a humidified 5%
CO2 incubator. Nonad-herent cells were removed
by washing twice with PBS. Adherent cells were identified by Giemsa staining. Only AM and bronchial epithelial cells
adhered to cell culture plates; neutrophils did not adhere and bronchial epithelial cells constituted less than 1% of adherent
cells. AM viability was measured by trypan blue exclusion.
Cell treatments AM that had been obtained from BAL fluid (BALF) were seeded into 24-well cell culture plates at a
concentration of 1×109 cells/L. First the cells were incubated for 1.5 h with either NAC (Sigma, St Louis, Missouri, USA) or
PDTC (Sigma, USA), and they were then stimulated by either
TNF-a (Biosea, Beijing, China) or IL-1 (Biosea, China). The AM
and supernatant were harvested, and the supernatant was stored at -80 °C until use.
Western blotting Cytoplasmic proteins were extracted from the cultured AM with NE-PER Nuclear and Cytoplasmic
Extraction Reagent (Pierce, Woburn, Massachusetts, USA) according to the manufacturer¡¯s instructions. The samples
containing 20 µg protein were incubated for 5 min in boiling water.
Prestained protein molecular weight markers (Fermentas,
Burlington, Ontario, Canada) were included in each gel.
Proteins were separated by 8% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), and then electroblotted onto Hybond-ECL nitrocellulose membrane. Non-specific
binding sites were blocked by immersing the membranes in Tris-buffered saline Tween (TBS-T) for 1 h at room temperature on
an orbital shaker. Membranes were then incubated in primary antibodies for 1 h at room temperature on an orbital shaker. The
membranes were then washed 5 times for 5 min each in TBS-T and incubated in the diluted peroxidase-conjugated immunopure
goat anti-rabbit IgG (H+L) (Pierce, USA) for 1 h at room temperature on an orbital shaker. After the membranes were again
washed 5 times for 5 min each in TBS-T, the proteins were visualized using enhanced chemiluminescence solution (ECL). The
images were developed on X-ray film and the band densities were analyzed by using a UVP-GDS8000 gel analysis system. As
a loading control, the same membranes were stripped and blotted with
anti-a-tubulin antibodies. The primary antibodies
were obtained from the following sources:
phospho-IkBa (Ser32) antibody, phospho-IKKalpha (Ser180)/IKKbeta (Ser181)
antibody from Cell Signaling Technology (Danvers, Massachusetts, USA); rabbit monoclonal antibody against human
a-tubulin fromSanta Cruz Biotechnology (Santa
Cruz, California, USA).
Electrophoretic mobility shift assay Nuclear protein was extracted from the cultured AM with NE-PER Nuclear and
Cytoplasmic Extraction Reagents (Pierce, USA) according to the manufacturer¡¯s instructions. The sequences of
biotin-NF-kB DNA probes that were double-strand end-labeled by biotin were 5¡¯-xGCCTGGGAAAGTCCCCTCAACT-3¡¯ (sense) and
5¡¯-xAGTTGAGGGGACTTTCCCAGGC-3¡¯ (anti-sense) (biotins are represented by x). The sequences of
Unlabeled-NF-kB DNA probes were 5¡¯-GCCTGGGAAAGT-CCCCTCAACT-3¡¯ (sense) and 5¡¯-AGTTGAGGGGACTTT-CCCAGGC-3¡¯ (antisense) (SBS
Biotechnology, Shanghai, China). NF-kB DNA binding activity was detected by using a LightShift Chemiluminescent
electrophoretic mobility shift assay (EMSA) kit(Pierce, USA) according to the manufac-turer¡¯s instructions as follows. A 5%
native polyacrylamide gel was prepared in 0.5×TBE (Tris-Borate EDTA), and pre-electrophoresed for 1 h. at 100 V in 0.5×TBE.
Binding reaction mixture containing 50 ng/µl Poly(dI·dC), 0.05% NP-40, 50 mmol/L KCl, 5 mmol/L
MgCl2 and 10 mmol/L ethylenediamine tetraacetic acid (EDTA) were incubated at room temperature for 20 min. Five microliters of 5× loading buffer
was added to each 20 µL of binding reaction mixture, mixed, and 20 µL of each sample was loaded onto the polyacrylamide gel.
Samples were electrophoresed at 100 V until the bromophenol blue dye had migrated approximately two-thirds to
three-quarters of the length of the gel, and then the binding reactions
wereelectroblotted ontonylon
membraneat 380 mA (~100 V) for 1 h. The membrane was cross-linked for 10 min at a distance of approximately 0.5 cm from an ultraviolet lamp equipped with
254 nm bulbs, and biotin-labeled DNA was visualized by using ECL. The images were developed on X-ray film and the band
densities were analyzed by using a UVP-GDS8000 gel analysis system (Ultra-Violet Products Ltd, Cambridge, UK).
Statistical analysis Data were analyzed with SPSS for Windows Version 11.5. All results are expressed as mean±SD.
Statistical evaluations of the data were performed with one-way analysis of variance (ANOV) and the paired-samples
t-test, and multiple comparisons in ANOV were analyzed by using the Student-Newman-Keuls test. For all tests,
P<0.05 was considered to be statistically significant.
Results
Effects of TNF-a and IL-1 on phosphorylation of
IkBa in COPD AM After stimulation with
TNF-a (10 ng/mL) or IL-1 (10 ng/mL), cytoplasmic proteins of the cultured AM were collected at various time points (0 min, 30 min, 60 min, 90 min, 120 min
and 150 min). The phosphorylation level of
IkBa increased in a time-dependent manner, and peak induction of the
phosphorylation was observed at 90 min (Figure 1A). To determine dose responsiveness, the cultured AM were incubated for 90 min
with TNF-a and IL-1 at concentrations from 0.1 ng/mL to 20 ng/mL. Both
TNF-a and IL-1 can induce the phosphorylation of
IkBa in a dose-dependent manner (P<0.05; Figure 1B).
Effect of NAC on phosphorylation of IKKa and
IKKb When AM were stimulated by TNF-a or IL-1 at a concentration of
10 ng/mL, the levels of both phospho-IKKb and
phospho-IKKa were significantly elevated (P<0.05; Figure 2). Our results
showed that NAC had no effect on the phosphorylation of
IKKb and IKKa induced by IL-1 (Figure 2B). Howerever, the
phosphorylation of IKKb and IKKa induced by TNF-a in the AM pretreated with NAC was significantly inhibited
(P<0.05; Figure 2A). The results suggest that the processes upstream of IKK activation induced by
TNF-a can be inhibited by NAC, which indirectly results in inhibition of the phosphorylation of
IKKb and IKKa.
Effects of NAC on phosphorylation of IkBa induced by
TNF-a and IL-1 NAC could inhibit the phosphorylation of
IkBa induced by TNF-a (Figure 3), but not affect the phosphorylation of
IkBa induced by IL-1 (Figure 3). Similarly, EMSA showed
that the activation of NF-kB induced by TNF-a was inhibited by NAC in a dose-dependent manner (Figure 4A), but that NAC
did not affect the activation of NF-kB induced by IL-1 (Figure 4B). Howerever, if 50 mmol/L NAC was directly added into the
nuclear extracts, NF-kB DNA binding activity was not directly inhibited by it
in vitro (Figure 5).
Effect of PDTC on the release of IkB AM were pretreated for 1.5 h with PDTC at concentrations ranging from 10 µmol/L
to 50 µmol/L, and then were stimulated for 90 min by
TNF-a or IL-1 at a concentration of 10 ng/mL. Cytoplasmic and
nuclear extracts were also prepared. Western blot analysis showed that PDTC does not inhibit the phosphorylation of
IkBa induced by TNF-a and IL-1 (Figure 6), whereas EMSA showed that the activation of
NF-kB induced by both TNF-a and IL-1 can be significantly inhibited by PDTC in a dose-dependent manner
(P<0.05; Figure 7). Howerever, PDTC (50 µmol/L) directly
added into the nuclear extracts, had no direct effect on
NF-kB DNA binding activity in vitro (Figure 5).
Discussion
In the classical NF-kB signaling pathway, upon stimulation by
TNF-a or IL-1, the IKK signalosome composed of
IKKa, IKKb, and IKKg is activated, which leads to the phosphorylation of
IkB on 2 conserved N-terminal serine residues. Then phosphorylated
IkB are ubiquitinated and subsequently degraded by the S26 proteasome. So
NF-kB is activated, and then translocated into the nucleus. Finally, it binds to a decameric consensus motif in target genes, resulting in their
transcription[6,17]. In the present study we found that the phosphorylation of
IKKa, IKKb, and IkBa induced by TNF-a can be inhibited by NAC. But we also found that NAC had no effect on the phosphorylation of
IKKa, IKKb, and IkBa induced by IL-1. Although the IKK responsible for
IkBa phosphorylation is the point of convergence for the activation of
NF-kB induced by either TNF-a or IL-1, there are different upstream mechanisms that result in IKK activation. Some studies have
indicated that activation of TNF receptor (TNFR) 1 triggered by
TNF-a can result in strong activation of IkB kinase (IKK) via
a pathway including TNFR1-associated protein with death domain (TRADD), TNFR-associated Factor (TRAF) 2,
receptor-interacting protein (RIP) and MAP kinase ERK kinase kinase 3
(MEKK3) [18_20]. Furthermore, upstream of IL-1-triggered IKK
activation, Toll/interleukin-1 receptor domain-containing adapter protein (TIRP), interleukin-1 receptor associated kinase 1
(IRAK1), IRAK4 and TRAF6 played important
roles[21,22]. So although NAC does not directly inhibit the phosphorylation of
IKKb and IKKa, it does inhibit the processes upstream of IKK activation induced by
TNF-a. The present study showed that NAC could inhibit the activation of
NF-kB induced by TNF-a, but not interfere with the activation of
NF-kB induced by IL-1. EMSA also showed that NAC didn¡¯t directly inhibit
NF-kB DNA binding activity in vitro. Therefore, we think that NAC
can inhibit the upstream of IKK activation induced by
TNF-a, which blocked TNF-a-stimulated NF-kB activation. We know
that the extracellular domain of TNFR is composed of 4 conserved cysteine-rich repeats, and 4_6 cysteines were involved in
disulfide bridges in the repeats [23]. Because NAC can cause structural changes in TNF receptors by reducing the disulfide
bridges, it can lower the affinity of TNFR1 or TRAF2, which is perhaps related to the action of NAC.
PDTC is a fairly specific inhibitor of NF-kB induction, which has no significant influence on the binding activity of oct-1
or proteins binding to a cAMP-response element and the GC-rich binding motif of
Spl[24]. Moreover, PDTC also blocks
NF-kB activation independently of the inducing agent and cell
line[24]. However, the nature of the pharmacological mechanism
by which it inhibits NF-kB activation remains controversial. In the present study, we found that PDTC had no effects on the
phosphorylation of IkBa induced by TNF-a or IL-1, but that the activation of
NF-kB induced by both TNF-a and IL-1 could be significantly inhibited by PDTC. We also found that PDTC does not directly inhibit
NF-kB DNA binding activity in vitro, which accords with the findings of Schreck
et al[24]. So it is evident that PDTC can prevent the degradation of
IkBa, and prevent the release of IkB from NF-kB, which results in the inhibition of
NF-kB activation. It has been shown that
ubiquitin-protein ligases (E3) play a key role in the ubiquitin-mediated
proteolytic cascade, conferring high specificity and selectivity
on the system[25,26]. Specific ring finger complexes (SCF complexes) belong to the ring finger domain-containing E3 family,
and SCFb-TrCP (transducin repeat-containing protein) is one member of the SCF complex. When
IkBa is phosphorylated on
Ser32 and Ser36, it will be recognized by
SCFb-TrCP, then ubiquitinated, and finally degraded by the 26S
proteasome[27_29]. So we speculate that PDTC can inhibit the activity of
SCFb-TrCP, which protects IkBa from being degraded via the
ubiquitylation-proteasome proteolytic pathway. Although signals that induce the phosphorylation of I¨ºB can also cause the
phosphorylation of NF-kB
proteins[30], recent evidence indicates that the phosphorylation of
NF-kB subunit p65 can modulate NF-kB transcription activity but not affect nuclear translocation or DNA
binding affinity[31,32]. This finding notwithstanding, it was
useful to further study whether PDTC can directly inhibit the phosphorylation of
NF-kB.
In short, although both NAC and PDTC are inhibitors of
NF-kB activation, the sites they inhibit are different, and their
function seems to have nothing to do with their antioxidant properties. These findings cast a new light on the treatment of
COPD on the basis of this new property of NAC.
Acknowledgement
We thank Sheng-dao XIONG, Jian-ping ZHAO, and Jin LIU for their technical assistance.
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