The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Krisanaprakornkit, S.
Right arrow Articles by Dale, B. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Krisanaprakornkit, S.
Right arrow Articles by Dale, B. A.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
The Journal of Immunology, 2002, 168: 316-324.
Copyright © 2002 by The American Association of Immunologists

Regulation of Human {beta}-Defensin-2 in Gingival Epithelial Cells: The Involvement of Mitogen-Activated Protein Kinase Pathways, But Not the NF-{kappa}B Transcription Factor Family1

Suttichai Krisanaprakornkit*, Janet R. Kimball* and Beverly A. Dale2,*,{dagger},{ddagger},§

Departments of * Oral Biology and {dagger} Periodontics, School of Dentistry, and Departments of {ddagger} Biochemistry and § Medicine/Dermatology, School of Medicine, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stratified epithelia of the oral cavity are continually exposed to bacterial challenge that is initially resisted by neutrophils and epithelial factors, including antimicrobial peptides of the {beta}-defensin family. Previous work has shown that multiple signaling pathways are involved in human {beta}-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-{kappa}B in hBD-2 regulation was investigated initially due to its importance in inflammation and infection. Nuclear translocation of p65 and NF-{kappa}B activation was seen in human gingival epithelial cells stimulated with F. nucleatum cell wall extract, indicating possible involvement of NF-{kappa}B in hBD-2 regulation. However, hBD-2 induction by F. nucleatum was not blocked by pretreatment with two NF-{kappa}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-{kappa}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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antimicrobial peptides of the {beta}-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 {beta}-defensins, human {beta}-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 {beta}-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{beta}, TNF-{alpha}, 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-{alpha} 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-{kappa}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-{kappa}B binding sequences (14). Nevertheless, the role of NF-{kappa}B in {beta}-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{beta} and TNF-{alpha}, 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-{kappa}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-{kappa}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-{kappa}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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The cell wall extract of F. nucleatum was prepared as described previously (8). PMA, cycloheximide (CHX), actinomycin D (ACT D), DMSO, and pyrrolidine dithiocarbamate (PDTC) were obtained from Sigma-Aldrich (St. Louis, MO). The proteasome inhibitor peptide-aldehyde MG132, SB203580, tyrphostin AG126, and AG9, an inactive analog of AG126, were obtained from Calbiochem (San Diego, CA). U0126 and PD98059 were purchased from Alexis Biochemicals (San Diego, CA). Polyclonal Abs specific for phosphorylated and nonphosphorylated forms of three MAP kinase pathways were purchased from Cell Signaling Technology (Beverly, MA). Control whole cell lysates and purified peptides for each specific signaling pathway were obtained from Cell Signaling Technology. mAb against p65 subunit of NF-{kappa}B and polyclonal Abs against p50 subunit of NF-{kappa}B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A consensus binding motif for NF-{kappa}B, the mutated binding motif, and specific TransCruz gel supershift Abs for five subunits of NF-{kappa}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-{kappa}B and its mutant binding motif (5 pmol each) were 5' end-labeled with 40 µCi of [{gamma}-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-{kappa}B or NF-{kappa}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-{kappa}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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nuclear translocation of NF-{kappa}B and binding to NF-{kappa}B consensus binding site are stimulated by F. nucleatum cell wall extract

We first examined possible participation of NF-{kappa}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-{kappa}B in hBD-2 mRNA up-regulation by F. nucleatum cell wall, we investigated the translocation of p65, a subunit of NF-{kappa}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. 1Go), suggesting the involvement of NF-{kappa}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. 2Go), indicating the presence of the heterodimeric complex of p65 and p50 in the nuclei. A transient increase in the p65 subunit of NF-{kappa}B was also observed after PMA activation.



View larger version (86K):
[in this window]
[in a new window]
  		FIGURE 1. Nuclear translocation of p65 subunit of NF-{kappa}B after HGE stimulation with F. nucleatum cell wall. HGE were grown on coverslips and stimulated with 10 µg/ml F. nucleatum cell wall for 1 h or left unstimulated (control). Cells were fixed in 4% paraformaldehyde and permeabilized with cold acetone, then reacted with mAb against p65 and FITC-conjugated (green) anti-mouse Ab. 4',6'-diamidino-2-phenylindole (DAPI)-stained DNA (blue) indicates the location of nuclei.

 


View larger version (71K):
[in this window]
[in a new window]
  		FIGURE 2. Activation of NF-{kappa}B in stimulated HGE. p65 and p50 subunits of NF-{kappa}B are increased in the nuclei of epithelial cells after stimulation with F. nucleatum cell wall. HGE were stimulated with 10 µg/ml F. nucleatum cell wall (F.n. CW) for various times. Western blots of nuclear protein extracts were analyzed with Abs specific for the p65 and p50 subunits of NF-{kappa}B. Nuclear extracts from Jurkat cells were used as a control. The polyclonal Ab against p50 used in this study also reacts with the precursor form of p50 subunit, p105. The nuclear extracts of HGE stimulated with 10 ng/ml PMA for various times were used as a control for the NF-{kappa}B activation in HGE, and the result showed the activation of p65 subunits of NF-{kappa}B within the short time after stimulation.

 
Gel shift assays were consistent with the findings from immunofluorescence and Western blot analyses. NF-{kappa}B bound to the NF-{kappa}B consensus DNA binding site in HGE stimulated with F. nucleatum cell wall and was effectively competed by cold probe (Fig. 3GoA). Gel supershift assay showed that only p65 and p50 subunits were present in the NF-{kappa}B-DNA binding complex (Fig. 3GoB). 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).



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 3. Activation of p65 and p50 subunits in F. nucleatum-stimulated HGE. A, The EMSA shows the DNA-NF-{kappa}B factor complex (arrow) in the nuclear extract from HGE stimulated with F. nucleatum cell wall (F.n. CW). Nuclear extracts (4 µg) from control HGE, HGE stimulated with 10 µg/ml F. nucleatum cell wall for 1 h, were incubated with 32P-labeled NF-{kappa}B or mutant probes. In some samples, the labeled probes competed with their respective cold oligonucleotide DNA in the ratio of 100:1 (cold:hot). There was no NF-{kappa}B-DNA binding complex in the nuclear protein sample reacted with mutant probe or competed with cold probe. B, The gel supershift assay shows that the p65 and p50 subunits (* and **, respectively) are the major subunits in the DNA-NF-{kappa}B complexes (arrow) from the nuclear extracts of HGE stimulated with 10 µg/ml F. nucleatum cell wall.

 
Two NF-{kappa}B inhibitors, PDTC and MG132, do not inhibit hBD-2 induction but block TNF-{alpha} induction, a known NF-{kappa}B target gene

To determine whether NF-{kappa}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. 4GoA or 6 h as shown in Fig. 4GoB). Surprisingly, hBD-2 induction was not blocked by any doses of PDTC and MG132 (Fig. 4Go, A and B), two commonly used NF-{kappa}B inhibitors that block the NF-{kappa}B pathway by different mechanisms. Pretreatment with MG132 alone regardless of stimulation induced hBD-2 mRNA (Fig. 4GoB; 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. 4GoB), suggesting that pretreatment with either MG132 or CHX may increase the stability of IL-8 mRNA.



View larger version (50K):
[in this window]
[in a new window]
  		FIGURE 4. Effect of inhibitors on hBD-2 mRNA induction. A, RPA. PDTC, an NF-{kappa}B inhibitor, does not inhibit hBD-2 induction by F. nucleatum cell wall. HGE were pretreated with PDTC (1–50 µM) for 1 h and then stimulated with 10 µg/ml F. nucleatum cell wall (F.n. CW) for 16 h. Total RNA was extracted and analyzed by RPA as previously described (9 ). Total RNA (20 µg) was probed with two different biotin-labeled RNA probes: hBD-2 and GAPDH, a housekeeping gene control. GAPDH is shown as a normalization control. B, hBD-2 induction by F. nucleatum cell wall is inhibited by ACT D and CHX, but not by two NF-{kappa}B inhibitors, PDTC and MG132. HGE were pretreated with either 1 µg/ml ACT D, 10 µg/ml CHX, 100 µM PDTC, 10 µM MG132, or DMSO (vehicle control) for 1 h, and then stimulated with 10 µg/ml F. nucleatum cell wall for 6 h, or left unstimulated. The short period of stimulation was chosen because longer stimulation in the presence of CHX and ACT D was toxic to the cells. Total RNA was extracted and analyzed by RT-PCR with specific primers for hBD-2, IL-8, and RPO (housekeeping gene). PCR for hBD-2 only was conducted for 30 cycles due to the lower levels of hBD-2 mRNA induction by stimulation with F. nucleatum cell wall for only 6 h; this resulted in a slightly increased background of hBD-2 mRNA in control unstimulated cells. Other PCRs were conducted for 25 cycles. Note that pretreatment with MG132 alone induced hBD-2 mRNA regardless of stimulant. C, MG132 inhibits TNF-{alpha} and PDTC shows dose-dependent inhibition of induction of TNF-{alpha}, an NF-{kappa}B target gene. HGE were pretreated with various doses of either MG132, PDTC, or DMSO for 1 h and stimulated with 10 µg/ml F. nucleatum cell wall for 1 h or left unstimulated. Total RNA was extracted and analyzed by RT-PCR with a specific primer pair for TNF-{alpha} (22 cycles). Relative expression of TNF-{alpha} in the presence of both doses of MG132 was ~30% of control; relative expression in the presence of PDTC was ~100, 40, and 22% for 10, 50, and 250 µM PDTC, respectively, as determined by densitometry.

 
The effect of PDTC and MG132 on up-regulation of TNF-{alpha}, a known target gene of NF-{kappa}B pathway (25, 26), was also tested in HGE. TNF-{alpha} was transiently and rapidly induced by F. nucleatum cell wall (9). However, induction of TNF-{alpha} mRNA was strongly inhibited by MG132 and was partially inhibited in a dose-dependent manner by PDTC (Fig. 4GoC), indicating the efficacy of these two inhibitors, especially MG132, in blocking the NF-{kappa}B pathway in this cell type.

To further investigate inhibition of the NF-{kappa}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. 5GoA), indicating that they are effective NF-{kappa}B pathway inhibitors in this cell type. This finding was confirmed by gel shift analysis (Fig. 5GoB). Because the p65 and p50 are the predominant pair of NF-{kappa}B family members in the HGE (Fig. 3GoB), these results imply that the NF-{kappa}B subunits are not required for hBD-2 regulation. In contrast, CHX, identified as a hBD-2 inhibitor (Fig. 4GoB), did not block nuclear translocation of p65 subunit and did not inhibit formation of the DNA-NF-{kappa}B binding complexes in the nuclear extracts of HGE stimulated with F. nucleatum cell wall (Fig. 5GoB). Similar to CHX, SB203580, identified as a hBD-2 inhibitor (Fig. 7GoB and Fig. 10, A and B), did not block the DNA-NF-{kappa}B complex (Fig. 5GoB). Taken together, these studies rule out the NF-{kappa}B transcription factor subunits as either necessary or sufficient for hBD-2 mRNA up-regulation in HGE.



View larger version (71K):
[in this window]
[in a new window]
  		FIGURE 5. Effects of inhibitors on NF-{kappa}B activation. A, MG132 and PDTC, but not CHX, block nuclear translocation of p65 subunit of NF-{kappa}B. HGE were grown on coverslips and pretreated with either 100 µM PDTC (C), 10 µM MG132 (E), 10 µg/ml CHX (D), or DMSO, vehicle control (F), for 1 h. HGE were then stimulated with 10 µg/ml F. nucleatum cell wall (B–F) for 1 h. Immunofluorescence was conducted as in Fig. 1Go. B, EMSA. MG132 and PDTC, but not CHX and SB203580, block the NF-{kappa}B-DNA binding complex (arrow) in the nucleus of HGE stimulated with F. nucleatum cell wall. HGE were pretreated with either 10 µM SB203580, 10 µM MG132, 100 µM PDTC, 10 µg/ml CHX, or DMSO for 1 h before stimulation with 10 µg/ml F. nucleatum cell wall for 1 h or left unstimulated (control). Nuclear protein extract (4 µg) was used in the binding reaction with 32P-labeled consensus NF-{kappa}B probe and resolved on a 5% polyacrylamide gel in 0.5x TBE (as in Fig. 3Go). Note the partial inhibition of NF-{kappa}B-DNA binding complex by pretreatment with PDTC and nearly complete inhibition with MG132.

 


View larger version (77K):
[in this window]
[in a new window]
  		FIGURE 7. Effects of MAP kinase inhibitors on hBD-2 mRNA induction by RT-PCR analysis. A, PD98059 and U0126, two selective inhibitors of MEK1/2 in the p44/42 MAP kinase pathway, inhibit hBD-2 induction by PMA but not by F. nucleatum cell wall. HGE were pretreated with either 20 µM PD98059, 20 µM U0126, 10 µM MG132, or DMSO, vehicle control, for 1 h, and then were stimulated with either 10 µg/ml F. nucleatum cell wall or 10 ng/ml PMA for 10 h. B, SB203580, an inhibitor of p38 MAP kinase, and tyrphostin AG126, an inhibitor of tyrosine phosphorylation of JNK, partially inhibit hBD-2 induction by F. nucleatum cell wall. HGE were pretreated with 1, 10, and 100 µM SB203580; 1, 10, and 100 µM AG126; a combination of 10 µM SB203580 and 1, 10, and 100 µM AG126; 100 µM AG9 (an inactive analog control for AG126); 10 µM MG132; 100 µM PDTC; or DMSO for 1 h. HGE were then stimulated with either 10 µg/ml F. nucleatum cell wall or 10 ng/ml PMA for 10 h. Total RNA was extracted and analyzed by RT-PCR (25–27 cycles). Note that the combination of SB203580 (10 µM) and AG126 (100 µM) completely inhibited hBD-2 induction by F. nucleatum cell wall (lane designated by arrow).

 
MAP kinase pathways are activated by phosphorylation in HGE stimulated with F. nucleatum cell wall

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. 6Go, A and B, respectively). However, F. nucleatum cell wall had little effect on the activation of p44/42 MAP kinase pathway (Fig. 6GoC). 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. 6GoC), 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. 6Go, 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. 6GoC). These results suggest the involvement of multiple MAP kinase pathways in hBD-2 regulation by different stimulants.



View larger version (80K):
[in this window]
[in a new window]
  		FIGURE 6. Activation of MAP kinase pathways in HGE. Two MAP kinase pathways, p38 (A) and JNK (B), are activated by phosphorylation after HGE are stimulated with F. nucleatum cell wall, while there is little effect on the activation of p44/42 (C). HGE were stimulated with either 10 µg/ml F. nucleatum cell wall or 10 ng/ml PMA for the indicated times. Whole cell lysate (20 µg) was resolved on 7.5–12% SDS-PAGE, blotted to nitrocellulose, and reacted with Abs specific for phosphorylated and nonphosphorylated forms of p38, JNK, and p44/42. Controls for each pathway were included in the analysis (two right lanes). Note that PMA slightly activates p44/42 MAP kinase (10 min after stimulation) (C), but not JNK (B) and p38 MAP kinases (A).

 
Inhibitors of MAP kinase pathways block hBD-2 induction by F. nucleatum cell wall and PMA

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. 7GoA), 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. 7GoA). 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. 7GoB). 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. 7GoB, 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. 8GoB). 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-{kappa}B, failed to block hBD-2 induction by both F. nucleatum cell wall and PMA (Fig. 7Go, 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. 7GoB). 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.



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 8. Effect of tyrphostin AG126 on phosphorylation of JNK MAP kinase. Tyrphostin AG126 inhibits activation of JNK MAP kinase (B), but not p38 (A) and p44/42 (C) MAP kinases, by F. nucleatum cell wall in a dose-dependent fashion. HGE were pretreated with 1, 10, and 100 µM AG126, 100 µM AG9, 10 µM MG132, or DMSO, for 1 h and then stimulated with 10 µg/ml F. nucleatum cell wall for 45 min. Whole cell proteins were extracted and 20 µg of whole cell lysates were used in Western blot analysis as described in Materials and Methods. C, Control unstimulated HGE. Note that the inactive AG9 had no effect and that MG132 enhanced phosphorylation of JNK MAP kinase.

 
The degree of hBD-2 induction by F. nucleatum cell wall and PMA and the percentage of hBD-2 inhibition by various doses of inhibitors were quantitatively analyzed by real-time PCR. Results showed dose-dependent inhibition of hBD-2 mRNA induction with F. nucleatum cell wall by SB203580 (~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. 9GoA). 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. 9GoB). The results of two independent experiments were very similar.



View larger version (50K):
[in this window]
[in a new window]
  		FIGURE 9. Real-time PCR assay of hBD-2 expression. A, hBD-2 induction by F. nucleatum cell wall is inhibited by SB203580 and tyrphostin AG126, as analyzed by real-time PCR. The bar graph shows hBD-2 induction relative to induction by F. nucleatum cell wall in the absence of inhibitors in two separate experiments: stippled bars, experiment 1; filled bars, experiment 2. All cDNA samples from Fig. 7GoB along with the known concentrations of hBD-2 and RPO plasmids were amplified in triplicate. The mean value of hBD-2 relative to the mean value of RPO in each sample was calculated and compared with the relative mean value from cDNA samples of F. nucleatum-stimulated cells (+F.n.) set at 100%. Note dose-dependent inhibition of hBD-2 induction by SB203580 and complete inhibition with 10 µM SB203580 and 100 µM tyrphostin AG126 combination. Little or no effect was observed with AG9, MG132, PDTC, and the DMSO vehicle control. B, hBD-2 induction by PMA is inhibited by SB203580 and tyrphostin AG126. The analysis was conducted as in A.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report several new findings on the regulation of hBD-2 mRNA in HGE in response to the commensal periodontal bacterium, F. nucleatum. First, our study clearly shows hBD-2 mRNA induction by a pathway that is independent of the NF-{kappa}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-{kappa}B, suggesting the possible involvement of NF-{kappa}B pathway, two NF-{kappa}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-{kappa}B family of transcription factors is not required, we cannot preclude involvement of upstream components of the NF-{kappa}B pathway that may also influence MAP kinase signaling.

The absence of NF-{kappa}B-dependent hBD-2 regulation

The proximal promoter of the hBD-2 gene has several consensus NF-{kappa}B binding sites, and NF-{kappa}B transcription factor is known to be activated in response to infection and inflammation. Furthermore, several studies have shown the role of NF-{kappa}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-{kappa}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-{kappa}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-{alpha}. In contrast, CHX and SB203580, which inhibit hBD-2 induction, do not block NF-{kappa}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-{alpha} (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-{kappa}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-{kappa}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. 7GoB). 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. 8GoB) 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-{kappa}B transcription factor family are not required for hBD-2 regulation under these conditions, although NF-{kappa}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.


   Acknowledgments
 
We thank Dr. Michael Dorschner for his expert assistance with real-time PCR and Robert Underwood for his help with immunofluorescence microscopy.


   Footnotes
 
1 This work was supported by National Institutes of Health, National Institute of Dental and Craniofacial Reasearch Grant DE 013573 and Comprehensive Center for Oral Health Research Grant P60 DE 97002. Back

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 Back

3 Abbreviations used in this paper: hBD, human {beta}-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. Back

Received for publication June 4, 2001. Accepted for publication October 25, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Weinberg, A., S. Krisanaprakornkit, B. A. Dale. 1998. Epithelial antimicrobial peptides: review and significance for oral applications. Crit. Rev. Oral Biol. Med. 9:399.[Abstract/Free Full Text]
  2. Scott, M. G., R. E. Hancock. 2000. Cationic antimicrobial peptides and their multifunctional role in the immune system. Crit. Rev. Immunol. 20:407.[Medline]
  3. Harder, J., J. Bartels, E. Christophers, J. M. Schröder. 2001. Isolation and characterization of human {beta}-defensin-3, a novel human inducible peptide antibiotic. J. Biol. Chem. 276:5707.[Abstract/Free Full Text]
  4. Yang, D., O. Chertov, S. N. Bykovskaia, Q. Chen, M. J. Buffo, J. Shogan, M. Anderson, J. M. Schröder, J. M. Wang, O. M. Howard, J. J. Oppenheim. 1999. {beta}-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286:525.[Abstract/Free Full Text]
  5. Tani, K., W. J. Murphy, O. Chertov, R. Salcedo, C. Y. Koh, I. Utsunomiya, S. Funakoshi, O. Asai, S. H. Herrmann, J. M. Wang, et al 2000. Defensins act as potent adjuvants that promote cellular and humoral immune response in mice to a lymphoma idiotype and carrier antigens. Int. Immunol. 12:691.[Abstract/Free Full Text]
  6. Ganz, T.. 1999. Defensins and host defense. Science 286:420.[Free Full Text]
  7. Huttner, K. M., C. L. Bevins. 1999. Antimicrobial peptides as mediators of epithelial host defense. Pediatr. Res. 45:785.[Medline]
  8. Krisanaprakornkit, S., A. Weinberg, C. N. Perez, B. A. Dale. 1998. Expression of the peptide antibiotic human {beta} defensin 1 in cultured gingival epithelial cells and gingival tissue. Infect. Immun. 66:4222.[Abstract/Free Full Text]
  9. Krisanaprakornkit, S., J. R. Kimball, A. Weinberg, R. P. Darveau, B. W. Bainbridge, B. A. Dale. 2000. Inducible expression of human {beta}-defensin-2 (hBD-2) by Fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and the role of commensal bacteria in innate immunity and the epithelial barrier. Infect. Immun. 68:2907.[Abstract/Free Full Text]
  10. Mathews, M., H. P. Jia, J. M. Guthmiller, G. Losh, S. Graham, G. K. Johnson, B. F. Tack, Jr. P.B. McCray. 1999. Production of {beta}-defensin antimicrobial peptides by the oral mucosa and salivary glands. Infect. Immun. 67:2740.[Abstract/Free Full Text]
  11. O’Neil, D. A., E. M. Porter, E. Elewaut, G. M. Anderson, L. Eckmann, T. Ganz, M. F. Kagnoff. 1999. Expression and regulation of the human {beta}-defensins hBD-1 and hBD-2 in intestinal epithelium. J. Immunol. 163:6718.[Abstract/Free Full Text]
  12. Becker, M. N., G. Diamond, M. W. Verghese, S. H. Randell. 2000. CD-1 dependent lipopolysaccharide-induced {beta}-defensin-2 expression in human tracheobronchial epithelium. J. Biol. Chem. 275:29731.[Abstract/Free Full Text]
  13. Kopp, E. B., S. Ghosh. 1995. NF-{kappa}B and Rel proteins in innate immunity. Adv. Immunol. 58:1.[Medline]
  14. Liu, L., L. Wang, H. P. Jia, C. Zhao, H. H. Q. Heng, B. C. Schutte, Jr P. B. McCray, T. Ganz. 1998. Structure and mapping of the human {beta}-defensin HBD-2 gene and its expression at sites of inflammation. Gene 222:237.[Medline]
  15. Tibbles, L. A., J. R. Woodgett. 1999. The stress-activated protein kinase pathways. Cell. Mol. Life Sci. 55:1230.[Medline]
  16. Ono, K., J. Han. 2000. The p38 signal transduction pathway: activation and function. Cell. Signal. 12:1.[Medline]
  17. Dale, B. A., J. R. Kimball, S. Krisanaprakornkit, F. A. Roberts, M. Robinovitch, R. O’Neal, E. Valore, T. Ganz, A. Weinberg. 2001. Localized antimicrobial peptide expression in human gingiva. J. Periodont. Res. 36:285.[Medline]
  18. Cobb, M. H., E. J. Goldsmith. 1995. How MAP kinases are regulated. J. Biol. Chem. 270:14843.[Free Full Text]
  19. Segar, R., E. G. Krebs. 1995. The MAPK signaling cascade. FASEB J. 9:726.[Abstract]
  20. Kyriakis, J. M., J. Avruch. 1996. Sounding the alarm: protein kinase cascades activated by stress and inflammation. J. Biol. Chem. 271:24313.[Free Full Text]
  21. Su, B., M. Karin. 1996. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr. Opin. Immunol. 8:402.[Medline]
  22. Aplin, A. E., R. L. Juliano. 1999. Integrin and cytoskeletal regulation of growth factor signaling to the MAP kinase pathway. J. Cell Sci. 112:695.[Abstract]
  23. Lee, K. A., M. R. Green. 1990. Small-scale preparation of extracts from radiolabeled cells efficient in pre-mRNA splicing. Methods Enzymol. 181:20.[Medline]
  24. Schreiber, E., P. Matthias, M. M. Müller, W. Schaffner. 1989. Rapid detection of octamer binding proteins with "mini-extracts," prepared from a small number of cells. Nucleic Acids Res. 15:6419.[Abstract/Free Full Text]
  25. Rojanasakul, Y., J. Ye, F. Chen, L. Wang, N. Cheng, V. Castranova, V. Vallyathan, X. Shi. 1999. Dependence of NF-{kappa}B activation and free radical generation on silica-induced TNF-{alpha} production in macrophages. Mol. Cell. Biochem. 200:119.[Medline]
  26. Ye, J., M. Ding, X. Zhang, Y. Rojanasakul, S. Nedospasov, V. Vallyathan, V. Castranova, X. Shi. 1999. Induction of TNF{alpha} in macrophages by vanadate is dependent on activation of transcription factor NF-{kappa}B and free radical reactions. Mol. Cell. Biochem. 198:193.[Medline]
  27. Hu, X., L. C. Moscinski, N. I. Valkov, A. B. Fisher, B. J. Hill, K. S. Zuckerman. 2000. Prolonged activation of the mitogen-activated protein kinase pathway is required for macrophage-like differentiation of a human myeloid leukemic cell line. Cell Growth Differ. 11:191.[Abstract/Free Full Text]
  28. Abe, K., H. Saito. 2000. The p44/42 mitogen-activated protein kinase cascade is involved in the induction and maintenance of astrocyte stellation mediated by protein kinase C. Neurosci. Res. 36:251.[Medline]
  29. Chaudhary, L. R., L. V. Avioli. 1997. Activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) by FGF-2 and PDGF-BB in normal human osteoblastic and bone marrow stromal cells: differences in mobility and in-gel renaturation of ERK1 in human, rat and mouse osteoblastic cells. Biochem. Biophys. Res. Commun. 238:134.[Medline]
  30. Novogrodsky, A., A. Vanichkin, M. Patya, A. Gazit, N. Osherov, A. Levitzki. 1994. Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors. Science 264:1319.[Abstract/Free Full Text]
  31. Diamond, G., V. Kaiser, J. Rhodes, J. P. Russell, C. L. Bevins. 2000. Transcriptional regulation of {beta}-defensin gene expression in tracheal epithelial cells. Infect. Immun. 68:113.[Abstract/Free Full Text]
  32. Bolstad, A. I., H. B. Jensen, V. Bakken. 1996. Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum. Clin. Microbiol. Rev. 9:55.[Abstract]
  33. Rogers, A. H.. 1998. Studies on fusobacteria associated with periodontal diseases. Aust. Dent. J. 43:105.[Medline]
  34. Konone, E.. 2000. Development of oral bacterial flora in young children. Ann. Med. 32:107.[Medline]
  35. Dean, J. L., R. Wait, K. R. Mahtni, G. Sully, A. R. Clark, J. Saklatvala. 2001. The 3' untranslated region of tumor necrosis factor {alpha} mRNA is a target of the mRNA-stabilizing factor HuR. Mol. Cell. Biol. 21:721.[Abstract/Free Full Text]
  36. Dixon, D. A., C. D. Kaplan, T. M. McIntyre, G. A. Zimmerman, S. M. Prescott. 2000. Post-transcriptional control of cyclooxygenase-2 gene expression: the role of the 3'-untranslated region. J. Biol. Chem. 275:11750.[Abstract/Free Full Text]
  37. Nakayama, K., A. Furusu, Q. Xu, T. Konta, M. Kitamura. 2001. Unexpected transcriptional induction of monocyte chemoattractant protein 1 by proteasome inhibition: involvement of the c-Jun N-terminal kinase-activator protein 1 pathway. J. Immunol. 167:1145.[Abstract/Free Full Text]
  38. Masaki, R., T. Saito, K. Yamada, R. Ohtani-Kaneko. 2000. Accumulation of phosphorylated neurofilaments and increase in apoptosis-specific protein and phosphorylated c-Jun induced by proteasome inhibitors. J. Neurosci. Res 62:75.[Medline]
  39. Rutberg, S. E., E. Saez, A. Glick, A. A. Dlugosz, B. M. Spiegelman, S. H. Yuspa. 1996. Differentiation of mouse keratinocytes is accompanied by PKC-dependent changes in AP-1 proteins. Oncogene 13:167.[Medline]
  40. Welter, J. F., J. F. Crish, C. Agarwal, R. L. Eckert. 1995. Fos-related antigen (Fra-1), junB, and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity. J. Biol. Chem. 270:12614.[Abstract/Free Full Text]
  41. Zhu, A. J., I. Haase, F. M. Watt. 1999. Signaling via {beta}1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro. Proc. Natl. Acad. Sci. USA 96:6728.[Abstract/Free Full Text]
  42. Vuong, H., T. Patterson, P. Shapiro, D. V. Kalvakolanu, R. Wu, W. Y. Ma, Z. Dong, S. R. Kleeberger, S. P. Reddy. 2000. Phorbol ester-induced expression of airway squamous cell differentiation marker, SPRR1B, is regulated by protein kinase C{delta}/Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway. J. Biol. Chem. 275:32250.[Abstract/Free Full Text]
  43. Takagi, Y., K. Nozaki, T. Sugino, I. Hattori, N. Hashimoto. 2000. Phosphorylation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase after transient forebrain ischemia in mice. Neurosci. Lett. 294:117.[Medline]
  44. Fleming, Y., C. G. Armstrong, N. Morrice, A. Paterson, M. Goedert, P. Cohen. 2000. Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7. Biochem. J. 352:145.
  45. Lian, J. P., R. Huang, D. Robinson, J. A. Badwey. 1999. Activation of p90RSK and cAMP response element binding protein in stimulated neutrophils: novel effects of the pyridinyl imidazole SB 203580 on activation of the extracellular signal-regulated kinase cascade. J. Immunol. 163:4527.[Abstract/Free Full Text]
  46. Minden, A., A. Lin, M. McMahon, C. Lange-Carter, B. Derijard, M. Cobb, R. J. Davis, G. L. Johnson, M. Karin. 1994. Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. Science 266:1719.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
C. Cosseau, D. A. Devine, E. Dullaghan, J. L. Gardy, A. Chikatamarla, S. Gellatly, L. L. Yu, J. Pistolic, R. Falsafi, J. Tagg, et al.
The Commensal Streptococcus salivarius K12 Downregulates the Innate Immune Responses of Human Epithelial Cells and Promotes Host-Microbe Homeostasis
Infect. Immun., September 1, 2008; 76(9): 4163 - 4175.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C.-Y. Kao, C. Kim, F. Huang, and R. Wu
Requirements for Two Proximal NF-{kappa}B Binding Sites and I{kappa}B-{zeta} in IL-17A-induced Human {beta}-Defensin 2 Expression by Conducting Airway Epithelium
J. Biol. Chem., May 30, 2008; 283(22): 15309 - 15318.
[Abstract] [Full Text] [PDF]