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-Defensin-2 in Gingival Epithelial Cells: The Involvement of Mitogen-Activated Protein Kinase Pathways, But Not the NF-
B Transcription Factor Family1
,
,
Departments of
*
Oral Biology and
Periodontics, School of Dentistry, and Departments of
Biochemistry and
Medicine/Dermatology, School of Medicine, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-defensin family. Previous work has shown that multiple signaling
pathways are involved in human -defensin (hBD)-2 mRNA regulation in
human gingival epithelial cells stimulated with a periodontal
bacterium, Fusobacterium nucleatum, and other
stimulants. The goal of this study was to further characterize these
pathways. The role of NF-
B in hBD-2 regulation was investigated
initially due to its importance in inflammation and infection. Nuclear
translocation of p65 and NF-B activation was seen in human gingival
epithelial cells stimulated with F. nucleatum cell wall
extract, indicating possible involvement of NF-B in hBD-2
regulation. However, hBD-2 induction by F. nucleatum was
not blocked by pretreatment with two NF-B inhibitors, pyrrolidine
dithiocarbamate and the proteasome inhibitor, MG132. To
investigate alternative modes of hBD-2 regulation, we explored
involvement of mitogen-activated protein kinase pathways. F.
nucleatum activated p38 and c-Jun
NH2-terminal kinase (JNK) pathways, whereas it had
little effect on p44/42. Furthermore, inhibition of p38 and JNK
partially blocked hBD-2 mRNA induction by F. nucleatum,
and the combination of two inhibitors completely blocked expression.
Our results suggest that NF-B is neither essential nor sufficient
for hBD-2 induction, and that hBD-2 regulation by F.
nucleatum is via p38 and JNK, while phorbol ester induces hBD-2
via the p44/42 extracellular signal-regulated kinase pathway. Studies
of hBD-2 regulation provide insight into how its expression may be
enhanced to control infection locally within the mucosa and thereby
reduce microbial invasion into the underlying
tissue. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-defensin family are expressed in all human
epithelial tissues tested to date and have been a subject of active
recent investigations. Their localization and characteristics support
the hypothesis that these peptides play a role in mucosal and skin
defense (1, 2). Three -defensins, human -defensin
(hBD)3-1, hBD-2, and
hBD-3, are expressed in human epithelial cells and skin keratinocytes.
hBD-1 and hBD-2 are both active against Gram-negative bacteria and have
more restricted activity against Gram-positive bacteria
(2). hBD-3 is active against Gram-positive bacteria,
having been isolated from skin using a strategy specifically designed
for this purpose (3). The proposed mechanism by which
these peptides function to kill bacteria is to form pores disrupting
bacterial membrane integrity. In addition to their direct antimicrobial
activity, it is now recognized that defensins have important signaling
potential, exhibiting cross-talk between the innate and acquired immune
responses. For example, hBD-2 stimulates dendritic cells via the CCR6
(4). Defensins may also act as adjuvants in enhancing Ab
production (5). Thus, these peptides have direct
antimicrobial properties and activate more long-term defenses
(6).
The interaction of microorganisms and epithelial cells is now
recognized to be an active process in which epithelial cells
participate in innate host defenses by expressing a variety of
proinflammatory cytokines, chemokines, and antimicrobial peptides,
including members of the -defensin family (7). In the
oral cavity, gingival epithelium surrounding a tooth forms a protective
barrier to separate environmental microorganisms from host tissues.
Human gingival epithelium is continually exposed to periodontal
microorganisms, such as the commensal Fusobacterium
nucleatum, and pathogenic Porphyromonas gingivalis,
that can lead to infections of gingival and periodontal tissues. In
cultured gingival epithelial cells, we and others previously showed the
constitutive expression of hBD-1 mRNA (8) and the
inducible expression of hBD-2 mRNA by cell wall extract of F.
nucleatum, IL-1, TNF-
, and PMA, an activator of epithelial
cells (9, 10). Further, we have shown the involvement of
multiple signaling pathways in hBD-2 regulation depending upon the type
of stimulant (9). hBD-3 mRNA was recently identified and
shown to be induced by TNF- and bacteria in cultured keratinocytes
and airway epithelial cells, suggesting that this hBD might be
important in the innate epithelial defense in skin and lung
(3). Evidence to date suggests that hBD-2 is up-regulated
via the NF-B transcription factor pathway in intestinal epithelial
(11) and tracheal epithelial cells (12). This
signaling pathway is critical for many cellular responses to
inflammatory stimuli (13), and the hBD-2 gene
(DEFB2) promoter region contains three potential
NF-B binding sequences (14). Nevertheless, the role of
NF-B in -defensin regulation is not fully understood, although it
is known to activate transcription of several genes involved in
inflammatory and immune responses.
We also examined the role of mitogen-activated protein (MAP) kinase
signaling pathways in hBD-2 regulation because of their importance in
cellular responses to stress, infection, inflammation, and cell growth
and differentiation (15, 16). The association of hBD-2
expression with differentiation in stratified gingival epithelium was
shown by immunolocalization in suprabasal cell layers of the tissue and
in differentiating keratinocytes in stimulated postconfluent cultures
(17). Three MAP kinase subfamilies have been identified in
mammalian cells: the extracellular signal-regulated kinase (ERK), the
c-Jun NH2-terminal kinase
(JNK)/stress-activated protein kinase, and the p38 MAP kinase
(18, 19, 20, 21). Environmental stress factors and proinflammatory
cytokines, including IL-1 and TNF-, preferentially activate JNK
and p38 MAP kinase pathways, whereas growth factors and phorbol ester
PMA activate the ERK pathway (18, 20, 21). In addition to
several binding sites for NF-B in the promoter region of the hBD-2
gene, there are several binding sites for AP-1, a downstream
transcription factor activated by three MAP kinase pathways. Therefore,
these signaling pathways, i.e., the NF-B pathway and three MAP
kinase pathways, are potential candidates for hBD-2 regulation in
gingival epithelial cells challenged by PMA or F. nucleatum
cell wall. In this study, we show that NF-B is neither critical nor
sufficient for hBD-2 regulation by F. nucleatum cell wall.
In contrast, our data suggest the involvement of distinct MAP kinase
pathways in hBD-2 regulation in response to different stimulants.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and polyclonal Abs against p50 subunit
of NF-B were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). A consensus binding motif for NF-B, the mutated binding motif,
and specific TransCruz gel supershift Abs for five subunits of NF-B
in the supershift assays were purchased from Santa Cruz
Biotechnology. General methods of epithelial cell culture, total RNA extraction, RNase protection assay (RPA), and RT-PCR analysis, and immunofluorescence for epithelial cells grown on coverslips were previously described (9).
Extraction of whole cell and nuclear proteins
Human gingival epithelial cells (HGE) were grown to 80% confluence before pretreatment with different doses of inhibitors (in some experiments) and then stimulated for various times with either 10 ng/ml PMA or 10 µg/ml F. nucleatum cell wall, previously identified as hBD-2 inducers (9). The F. nucleatum preparation was previously used between 1 and 50 µg/ml for up to 24 h with no apparent damage to the cells. The concentration tested here was useful for the short time intervals necessary to examine early events of hBD-2 regulation. Whole cell lysates were extracted in keratinocyte extraction buffer (22) containing 50 mM HEPES (pH 7.5), 1% Nonidet P-40 (Sigma-Aldrich), 0.5% sodium deoxycholate (Sigma-Aldrich), 50 mM NaCl, 50 mM NaF, 1 mM sodium orthovanadate (Sigma-Aldrich), 1 mM nitrophenylphosphate, 10 µg/ml aprotinin (Sigma-Aldrich), 5 mM benzamidine (Sigma-Aldrich), and 2 mM PMSF (Calbiochem). The lysates were then transferred to 1.5-ml centrifuge tubes, vortexed vigorously, and centrifuged for 5 min at 4°C. Protein content in the supernatant was determined by protein assay (Bio-Rad, Hercules, CA) with gammaglobulin as a standard. The method of nuclear protein extraction was modified from Lee and Green (23) and Schreiber et al. (24). Briefly, epithelial cells were trypsinized with trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA; Life Technologies, Rockville, MD), and then pelleted and washed with PBS. The cell pellet was resuspended in 1 ml of cold buffer A, containing 10 mM HEPES (pH 8), 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT (Bio-Rad), 1 µg/ml pepstatin (Boehringer Mannheim, Indianapolis, IN), 1 µg/ml leupeptin (Boehringer Mannheim), and 0.5 mM PMSF. Cells were allowed to swell on ice for 15 min and then were lysed with 200 µl of 10% Nonidet P-40 (Sigma-Aldrich). Nuclear pellets were isolated from the whole cell protein by centrifugation at 14,000 rpm for 30 s at 4°C and resuspended in two-thirds packed cell volume of cold buffer C with vigorous agitation in the cold room for 30 min. The buffer C contains 20 mM HEPES (pH 8), 1.5 mM MgCl2, 25% glycerol (v/v), 420 mM NaCl, 0.2 mM EDTA (pH 8), 1 mM DTT, 0.5 mM PMSF, 1 µg/ml pepstatin, and 1 µg/ml leupeptin. Nuclear debris was pelleted in the cold, leaving nuclear protein in the supernatant. Protein concentration of each nuclear extract was determined and aliquots were snap-frozen with ethanol and dry ice, stored at -70°C, and subsequently analyzed by Western blot hybridization. For the EMSA and gel supershift assay, the nuclear protein was dialyzed using a Slide-A-lyzer dialysis cassette MWCO 7000 (Pierce, Rockford, IL) for 2 h in the cold room against buffer D, containing 20 mM HEPES (pH 8), 20% glycerol (v/v), 100 mM KCl, 0.2 mM EDTA (pH 8), 1 mM DTT, 0.5 mM PMSF, 1 µg/ml pepstatin, and 1 µg/ml leupeptin. After dialysis, each nuclear extract was assayed for its protein content and aliquots were stored at -70°C.
Western blot analysis
Whole cell lysates and nuclear proteins as well as their controls were resolved on a 7.5–12% gradient SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) for 12 h in the cold. The membranes were blocked and incubated with primary Ab specific for each protein, diluted in appropriate buffer according to the manufacturer’s recommendation. The membranes were then incubated with HRP-conjugated secondary Ab (New England Biolabs) at 1/2000 in 10 ml of blocking buffer for 1 h at room temperature with gentle agitation, and then incubated with ECL substrates (0.5 ml of 20x LumiGlo and 0.5 ml of 20x peroxidase; Cell Signaling Technology) for 1 min at room temperature. The excess developing solution was drained off the membranes, and the membranes were subsequently exposed to Biomax ML film (Kodak, Rochester, NY) for various times depending on the intensity of the signals.
EMSA and gel supershift assay
A consensus binding motif of NF-B and its mutant binding
motif (5 pmol each) were 5' end-labeled with 40 µCi of
[
-32P]dATP by 20 U of T4 polynucleotide
kinase (New England Biolabs) in a total volume of 50 µl at 37°C for
1 h. Labeled oligonucleotide probes were purified from
unincorporated radioactive 32P by Microspin G-25
columns (Amersham Pharmacia Biotech, Piscataway, NJ). Binding reactions
(in a total volume of 20 µl) were performed by preincubating 4 µg
of nuclear extract protein in 20 mM HEPES (pH 7.9), 50 mM KCl, 0.5 mM
DTT, 0.2 mM EDTA, 1 mM MgCl2, 4% Ficoll, and 4
µg of poly(dI-dC) (Sigma-Aldrich) for 10 min at room temperature.
Subsequently, 5 x 104 cpm (
0.2
ng) of the double-stranded 32P-labeled probe
(either NF-B or NF-B mutant) was added and the mixture was
incubated on ice for 30 min. After incubation, 2 µl of loading buffer
(0.25% bromophenol blue in 40% glycerol) was added, and the samples
were run immediately on nondenaturing 5% polyacrylamide gels (19:1
acrylamide:bisacrylamide). The samples were resolved in running buffer
0.5x TBE (45 mM Tris base, 45 mM boric acid, and 1 mM EDTA) at 150
volts at 4°C. Subsequently, gels were dried at 60°C and exposed to
the X-OMAT AR films (Kodak) at -70°C between intensifying screens.
Competitions were performed by using unlabeled double-stranded
oligonucleotides to compete with the labeled oligonucleotide probe in
the ratio of 100:1 (20:0.2 ng). For gel supershift assays, 1 µl of
each specific Ab was added into the mixture after incubation with the
NF-B probe. The reaction was done at room temperature for 1 h.
The samples were then loaded immediately onto the gel as described
above.
Preparation of plasmid vectors for RPO and hBD-2
Inserts for ribosomal phosphoprotein (RPO) and hBD-2 were prepared by PCR of cDNA using a specific primer pair for RPO (forward primer, 5'-AGCAGGTGTTCGACAATGGCA-3'; and reverse primer, 5'-ACTCTTCCTTGGCTTCAACCT-3'), hBD-2 (forward primer, 5'-GGTGAAGCTCCCAGCCATCAG-3'; and reverse primer, 5'-CATCTTTGGACACCATAGTTT-3'). The PCR products were purified by a QIAquick PCR purification column (Qiagen, Valencia, CA) and inserted into the pCR II-TOPO plasmid (Invitrogen, Carlsbad, CA). The plasmids were then transformed into chemically competent TOP10F' Escherichia coli. Transformed clones were picked based on the white-blue screening and verified for an insert by EcoRI (Promega, Madison, WI) restriction digestion. Each clone was expanded by inoculating the transformed bacteria in 250 ml of Luria-Bertani broth, and plasmid vectors were isolated with the CONCERT High Purity Plasmid Maxiprep system (Life Technologies). The concentration of each plasmid was determined by UV absorbance and diluted to nanogram levels to be used as standards for real-time PCR analyses.
Real-time PCR
cDNA prepared from 3 µg of total RNA was analyzed using the LightCycler-FastStart DNA Master SYBR Green I system (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer’s protocol. The FastStart reaction mix consisted of reaction buffer, 10 µM MgCl2, dNTP mix (with dUTP instead of dTTP), SYBR green I dye, and Taq polymerase. A 17-µl aliquot of the reaction mixture, which included 2 µl of the FastStart reaction mix, 1 µl of each forward and reverse primer for either RPO or hBD-2 at 0.5 µM, 2.5 mM MgCl2 (final concentration), and water, was transferred to the LightCycler glass capillaries, and 3 µl of cDNA samples were then added to each tube. The temperature profile consisted of denaturation at 95°C for 10 min followed by 40 cycles of amplification: denaturation at 95°C, annealing at 65°C for 10 s, and extension at 72°C for 15 s, with a temperature transition rate of 20°C/s. After completing 40 cycles of PCR, the temperature was gradually increased to 95°C at a rate of 0.1°C/s to determine the melting temperature of PCR products. The purity of amplified product (either RPO or hBD-2) was determined as a single peak of melting curve without any small peaks of lower melting temperature from primer dimerization. The noise band was set to 0.1, which corresponded to the final fluorescence intensity of the negative sample, water. After background subtraction, the fluorescence of SYBR green dye was measured at 530 nm during the extension phase. The amount of hBD-2 or RPO mRNA in each sample was determined from the fluorescence intensity during the increasing phase of amplification in comparison to the fluorescence intensities of the known concentrations of standard hBD-2 or RPO plasmid. Real-time PCR was conducted in triplicate for each sample, and the mean value was calculated. The relative ratio of hBD-2 to RPO in each sample was calculated and compared with the relative ratio of F. nucleatum- or PMA-stimulated sample set at 100%. This procedure was performed in two independent experiments. From amplification plots using a series of three-fold dilutions of hBD-2 or RPO plasmid, we obtained a linear correlation between cycle number and the log amount of template at 1–300 ng for RPO plasmid and 0.3–100 ng for hBD-2 plasmid (r = -0.99) (data not shown).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and binding to NF-B consensus
binding site are stimulated by F. nucleatum cell wall
extract
We first examined possible participation of NF-B in hBD-2 mRNA
regulation by exposing HGE to F. nucleatum cell wall
extract. Purified F. nucleatum LPS, anticipated to be the
active component, was previously shown to be a poor stimulant
(9), and the identity of the active stimulant(s) is not
yet known. Use of this bacterial cell wall extract is biologically
relevant because this commensal organism is found within the restricted
space of the gingival sulcus where the epithelium is exposed to both
intact bacteria and bacterial debris.
To examine participation of NF-B in hBD-2 mRNA up-regulation by
F. nucleatum cell wall, we investigated the translocation of
p65, a subunit of NF-B, into the nucleus by immunofluorescence. HGE
were grown on coverslips and then either left unstimulated or
stimulated with F. nucleatum cell wall. While p65 subunit
localized in the cytoplasm of unstimulated control HGE, p65
translocated into the nucleus of F. nucleatum-stimulated HGE
(Fig. 1
), suggesting the involvement of
NF-B in cellular responses to F. nucleatum cell wall.
Moreover, the levels of both p65 and p50 subunits were transiently
increased in the nuclear extracts of HGE stimulated with F.
nucleatum cell wall within the first few hours (Fig. 2), indicating the presence of the
heterodimeric complex of p65 and p50 in the nuclei. A transient
increase in the p65 subunit of NF-B was also observed after PMA
activation.
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B bound to the
NF-B consensus DNA binding site in HGE stimulated with F.
nucleatum cell wall and was effectively competed by cold probe
(Fig. 3A). Gel supershift
assay showed that only p65 and p50 subunits were present in the
NF-B-DNA binding complex (Fig. 3B). Other subunits,
including Rel B, c-Rel, and p52, were not significantly represented in
this complex, consistent with the results from immunoblot analysis with
specific Ab against each subunit (data not shown).
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B inhibitors, PDTC and MG132, do not inhibit hBD-2
induction but block TNF- induction, a known NF-B target gene
To determine whether NF-B is critical for hBD-2 mRNA
up-regulation by F. nucleatum cell wall, HGE were pretreated
with various doses of PDTC, MG132, or DMSO, a vehicle control, and then
stimulated with F. nucleatum cell wall for indicated times
(16 h as shown in Fig. 4A or
6 h as shown in Fig. 4B). Surprisingly, hBD-2 induction
was not blocked by any doses of PDTC and MG132 (Fig. 4, A
and B), two commonly used NF-B inhibitors that block the
NF-B pathway by different mechanisms. Pretreatment with MG132 alone
regardless of stimulation induced hBD-2 mRNA (Fig. 4B; also
see Fig. 10A). The lack of hBD-2 inhibition by MG132 in HGE
differs from results in an intestinal epithelial cell line in which
hBD-2 induction was inhibited by MG132 (11), suggesting a
difference in the regulatory pathways of primary gingival epithelial
cells and immortalized epithelial cell lines (HT-29 and Caco cells).
Consistent with previous results (9), hBD-2 mRNA induction
was inhibited by ACT D and CHX, suggesting that hBD-2 induction
requires both new gene transcription and new protein synthesis.
Pretreatment with MG132 alone induced IL-8 mRNA, and treatment with
both MG132 and F. nucleatum cell wall induced IL-8 mRNA more
than treatment with F. nucleatum cell wall alone
(Fig. 4B), suggesting that pretreatment with either MG132 or
CHX may increase the stability of IL-8 mRNA.
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, a known
target gene of NF-B pathway (25, 26), was also tested
in HGE. TNF- was transiently and rapidly induced by F.
nucleatum cell wall (9). However, induction of
TNF- mRNA was strongly inhibited by MG132 and was partially
inhibited in a dose-dependent manner by PDTC (Fig. 4
C),
indicating the efficacy of these two inhibitors, especially MG132, in
blocking the NF-B pathway in this cell type.
To further investigate inhibition of the NF-B pathway in HGE,
cultures were pretreated with MG132 and PDTC, as well as with CHX, and
then stimulated with F. nucleatum cell wall.
Immunofluorescence showed that PDTC and MG132 blocked nuclear
translocation of p65 subunit in response to F. nucleatum
cell wall (Fig. 5A),
indicating that they are effective NF-B pathway inhibitors in this
cell type. This finding was confirmed by gel shift analysis (Fig. 5B). Because the p65 and p50 are the predominant pair of
NF-B family members in the HGE (Fig. 3B), these results
imply that the NF-B subunits are not required for hBD-2 regulation.
In contrast, CHX, identified as a hBD-2 inhibitor (Fig. 4B),
did not block nuclear translocation of p65 subunit and did not inhibit
formation of the DNA-NF-B binding complexes in the nuclear extracts
of HGE stimulated with F. nucleatum cell wall (Fig. 5B). Similar to CHX, SB203580, identified as a hBD-2
inhibitor (Fig. 7B and Fig. 10, A and
B), did not block the DNA-NF-B complex (Fig. 5B). Taken together, these studies rule out the NF-B
transcription factor subunits as either necessary or sufficient for
hBD-2 mRNA up-regulation in HGE.
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To examine whether MAP kinase signaling pathways are involved in
hBD-2 regulation by F. nucleatum cell wall, HGE were
stimulated with F. nucleatum cell wall for various times and
total cellular proteins were extracted. Western blot analysis with
specific Abs against each MAP kinase pathway showed the rapid and
transient activation of p38 MAP kinase (5–60 min) and JNK MAP kinase
(15–60 min) pathways (Fig. 6, A and B, respectively). However, F.
nucleatum cell wall had little effect on the activation of p44/42
MAP kinase pathway (Fig. 6C). Control cell lysates and
purified p42 were included in the analysis to show specific recognition
of the phosphorylated form of each MAP kinase pathway by its Ab. The
p44/42 MAP kinase was already activated in control unstimulated HGE
(Fig. 6C), probably due to the use of epidermal growth
factor to promote proliferation of keratinocytes in the keratinocyte
growth medium. While F. nucleatum cell wall preferentially
activated p38 and JNK MAP kinase pathways, PMA did not (Fig. 6, A and B). The absence of p38 and JNK activation
by PMA during 3 h after stimulation may be consistent with the
slow activation of hBD-2 mRNA by PMA (9). PMA slightly
activated p44/42 MAP kinase pathway in HGE at 10 min after stimulation
(Fig. 6C). These results suggest the involvement of multiple
MAP kinase pathways in hBD-2 regulation by different stimulants.
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To determine whether MAP kinase pathways are involved in hBD-2
up-regulation by both F. nucleatum cell wall and PMA, HGE
were pretreated with various inhibitors before stimulation with either
F. nucleatum cell wall or PMA. Pretreatment with PD98059 and
U0126, two selective inhibitors of MAP/ERK kinase (MEK)1/2 in the
p44/42 MAP kinase pathway, partially or totally blocked hBD-2 induction
by PMA, respectively (Fig. 7A), consistent with the known
effect of PMA on the activation of p44/42 MAP kinase pathway in other
cell types, e.g., myeloid cells, cortical astrocytes, primary
osteoblastic cells, etc. (27, 28, 29). However, hBD-2
induction by F. nucleatum cell wall was not blocked by these
two inhibitors (Fig. 7A). Pretreatment of HGE with various
doses of SB203580, a specific inhibitor of p38 MAP kinase at the
concentration 
10 µM, and tyrphostin AG126, reported to inhibit
tyrosine phosphorylation of p42 MAP kinase by LPS in murine macrophages
(30), partially blocked hBD-2 induction by F.
nucleatum cell wall and completely blocked hBD-2 induction by PMA
(Fig. 7B). The combination of SB203580 and tyrphostin AG126
(1, 10, and 100 µM) additionally inhibited hBD-2 induction by
F. nucleatum cell wall more than tyrphostin AG126 alone. The
inhibition was dose dependent with complete blocking by 10 µM
SB203580 and 100 µM tyrphostin AG126 (Fig. 7B, lane
designated by an arrow). Although tyrphostin AG126 is an inhibitor of
p42 MAP kinase in some cell types (30), in the HGE,
tyrphostin AG126 inhibited the activation of the JNK MAP kinase pathway
to a greater extent than either the p38 or p42/44 MAP kinase pathways
(Fig. 8B). Tyrphostin AG9, an
inactive analog of tyrphostin AG126, showed no inhibitory effect on
hBD-2 induction. Unlike all of the inhibitors of MAP kinase pathways,
PDTC and MG132, inhibitors of NF-B, failed to block hBD-2 induction
by both F. nucleatum cell wall and PMA (Fig. 7, A
and B). Induction of IL-8 mRNA by F. nucleatum
cell wall was also partially inhibited by SB203580 and tyrphostin AG126
and the combination of these two inhibitors in a dose-dependent
fashion, indicating the efficacy of these two inhibitors in blocking
cellular activation (Fig. 7B). Taken together, these
findings suggest that JNK and p38 MAP kinase pathways are critical for
hBD-2 up-regulation by F. nucleatum cell wall, whereas hBD-2
induction by PMA is via the activation of p44/42 MAP kinase
pathway.
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70, 86.5, and 98% for 1, 10,
and 100 µM, respectively), by 100 µM tyrphostin AG126 (93%),
and by combination of 10 µM SB203580 and tyrphostin AG126 (89, 95,
and 100% for 1, 10, and 100 µM, respectively) (Fig. 9A). Moreover, the level of
hBD-2 mRNA induced by PMA was inhibited by 10 µM SB203580 (90%)
and by 100 µM tyrphostin AG126 (98%) (Fig. 9B). The
results of two independent experiments were very similar.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B transcription
factor family. Although we show that exposure to F.
nucleatum cell wall results in rapid and transient activation of
p65 and p50 subunits of NF-B, suggesting the possible involvement of
NF-B pathway, two NF-B inhibitors, PDTC and MG132, fail to block
hBD-2 induction. Second, our study suggests that MAP kinase pathways
are necessary and may be sufficient for hBD-2 regulation in HGE in
response to both the bacterial stimulant and PMA. Using several
inhibitors that block different MAP kinase pathways, we show that hBD-2
induction is inhibited completely or partially depending on the doses
of inhibitors and the type of stimulants. Third, hBD-2 mRNA regulation
is mediated by multiple signaling pathways depending upon whether the
stimulation is from bacterial or chemical stimulants. This was
initially suggested by comparing the kinetics of hBD-2 up-regulation
using F. nucleatum cell wall and PMA as stimulants
(9) and is confirmed in this study by the differential
activation of signaling pathways. F. nucleatum cell wall
preferentially activates both JNK and p38 MAP kinase pathways, while it
has little effect on p44/42 MAP kinase pathways during the first 3
h of stimulation. In contrast, PMA does not activate JNK or p38 MAP
kinase pathways. Finally, while these studies suggest that the NF-B
family of transcription factors is not required, we cannot preclude
involvement of upstream components of the NF-B pathway that may also
influence MAP kinase signaling.
The absence of NF-B-dependent hBD-2 regulation
The proximal promoter of the hBD-2 gene has several consensus
NF-B binding sites, and NF-B transcription factor is known to be
activated in response to infection and inflammation. Furthermore,
several studies have shown the role of NF-B in hBD-2 regulation in
other cell types, including an intestinal epithelial cell line
(11), and in tracheobronchial epithelial cells
(12), as well as for the homologous tracheal antimicrobial
peptide in bovine tracheal epithelial cells (31). However,
the two NF-B inhibitors used in this study failed to block hBD-2
induction in HGE, and one of these inhibitors, MG132, resulted in
superinduction of hBD-2 mRNA. Several important differences could be
responsible for these contrasting findings in different cell types.
First, our study used primary epithelial cells from a stratified
epithelial tissue rather than cells from a nonstratified epithelium or
from immortalized cell lines (11, 12, 31). Second, we used
a bacterial stimulant, not a cytokine. F. nucleatum is a
Gram-negative oral commensal bacterium. Although it may be pathogenic
in some sites (32), it is not associated with periodontal
disease (33, 34). Our findings suggest the possibility
that commensal organisms may activate epithelial cells via a mechanism
that does not result in up-regulation of a whole battery of genes
associated with innate immune responses, but rather uses a mechanism
that leads to a discrete and limited response. Gingival epithelium is
constantly exposed to a variety of microorganisms from dental plaque,
and even healthy, noninflamed tissue is already activated to express
hBD-2 (17), in contrast to epidermis (14) or
intestine (11). This heightened state of readiness may
contribute to the protective epithelial barrier function of gingival
epithelium (9, 17).
To demonstrate the efficacy of the two NF-B inhibitors in HGE, we
showed that they block nuclear translocation and function in DNA
binding as well as inhibiting up-regulation of a known target gene,
TNF-. In contrast, CHX and SB203580, which inhibit hBD-2 induction,
do not block NF-B activation. Inhibition of hBD-2 mRNA induction by
CHX suggests that hBD-2 induction requires new protein synthesis, and
that some intermediate signal, possibly a cytokine or one or more
transcription factors necessary for hBD-2 induction, must be newly
synthesized. The observation that MG132, a proteasome inhibitor that
causes accumulation of ubiquitinated proteins, results in
superinduction of hBD-2 mRNA is also consistent with this hypothesis.
MG132 enhances the stability of short-lived proteins, including
proinflammatory cytokines such as TNF- (35) and
cyclooxygenase-2 (36) as well as transcription
factors, e.g., c-jun (37, 38).
Involvement of MAP kinase pathways in hBD-2 regulation
These surprising results with NF-B and hBD-2 inhibitors
prompted us to investigate the role of other signaling pathways in
hBD-2 regulation. We previously demonstrated an association between
hBD-2 expression and differentiation (17). The phorbol
ester, PMA, is a factor that induces hBD-2 mRNA (9) and
stimulates differentiation of epithelial cells via activation of
protein kinase C, MAP kinase pathways, and AP-1 (39, 40, 41, 42).
Because the promoter region of the hBD-2 gene contains several AP-1
binding sites as well as NF-B binding sites (14), we
examined the role of MAP kinase pathways, activators of the AP-1
transcription factor family that is involved in the control of cell
proliferation, maturation, and differentiation in many cell types,
including keratinocytes (36, 37, 38, 39). Both by the kinetics of
activation and by inhibitor studies, our evidence shows involvement of
MAP kinase pathways in hBD-2 induction in this cell type. Our findings
suggest that F. nucleatum cell wall stimulates gingival
epithelial cells by activating JNK and p38 MAP kinases with little
effect on p44/42 MAP kinase. The activation of JNK and p38 MAP kinases
may then regulate gene transcription of hBD-2 through transcription
factor AP-1. Furthermore, the specific p38 MAP kinase inhibitor,
SB203580, and the inhibitor, tyrphostin AG126, which preferentially
affects the JNK pathway in HGE, both partially inhibit hBD-2 induction,
and combination of these two inhibitors completely blocks hBD-2
induction by F. nucleatum cell wall. The most direct
interpretation of our results is that simultaneous activation of JNK
and p38 MAP kinase pathways by F. nucleatum cell wall
results either directly or indirectly in hBD-2 up-regulation. It is
commonly found that these pathways work together and are simultaneously
activated by stress-related conditions in many cell types (43, 44). In contrast, PMA does not activate JNK and p38 MAP kinases;
PMA effects on hBD-2 induction are blocked by the MEK1/2 inhibitors,
PD98059 and U0126, suggesting that hBD-2 mRNA induction by PMA is via
p44/42 MAP kinase pathway. Pretreatment with SB203580 at concentrations
greater than 10 µM has been reported to inhibit p44/42 MAP kinase in
neutrophils (45), and this may be why pretreatment with
SB203580 inhibits hBD-2 induction by PMA (Fig. 7B).
Activated p44/42 MAP kinase can phosphorylate and increase the activity
of transcription factor T cell factor/Elk-1, which is required
for enhanced transcription of the c-fos gene
(46). Activated JNK MAP kinase phosphorylates c-Jun at two
critical N-terminal residues, Ser63 and
Ser73, which enhances the ability of c-Jun to
activate transcription (21, 46). The final effect of the
two stimulants, F. nucleatum cell wall and PMA, which
activate hBD-2 via different MAP kinase pathways, could lead to
increased AP-1 activity. Superinduction of hBD-2 mRNA by MG132 also
fits this model for a role of JNK in hBD-2 regulation; activation of
JNK and phosphorylation of c-jun were found in response to
MG132 in PC12 cells and mesangial cells (37, 38). In our
evaluation of the JNK pathway, MG132 treatment results in an increase
in phosphorylated forms of JNK (Fig. 8B) and
c-jun (not shown), suggesting that proteasome inhibition may
also activate the JNK pathway in HGE cells.
In conclusion, our data implicate a role for MAP kinase pathways
possibly involving the AP1 transcription factor family, in hBD-2 mRNA
regulation in HGE in response to both a commensal bacterial stimulant
and PMA. We also suggest that members of the NF-B transcription
factor family are not required for hBD-2 regulation under these
conditions, although NF-B may be involved in hBD-2 regulation in
other epithelial cell types, or in the presence of other stimulants.
Regulation of hBD peptides is complex, but studies of regulation are
important to provide better understanding of molecular mechanisms and
approaches that could enhance the expression of these peptides for
future therapeutic uses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Beverly A. Dale, Department of Oral Biology, School of Dentistry, University of Washington, Box 357132, Seattle, WA 98195-7132. E-mail address: bdale{at}u.washington.edu
3 Abbreviations used in this paper: hBD, human -defensin; ACT D, actinomycin D; CHX, cycloheximide; HGE, human gingival epithelial cell; MAP, mitogen-activated protein; PDTC, pyrrolidine dithiocarbamate; RPA, RNase protection assay; RPO, ribosomal phosphoprotein; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; MEK, MAP/ERK kinase.
Received for publication June 4, 2001. Accepted for publication October 25, 2001.
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