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International Immunology Advance Access published online on November 6, 2007
International Immunology, doi:10.1093/intimm/dxm115
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Involvement of phospholipase D in regulating expression of anti-microbial peptide human ß-defensin-2

Suttichai Krisanaprakornkit1, Pareena Chotjumlong2, Prachya Kongtawelert2 and Vichai Reutrakul3

1 Department of Odontology and Oral Pathology, Faculty of Dentistry
2 Thailand Excellence Center for Tissue Engineering, Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand
3 Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

Correspondence to: Correspondence to: V. Reutrakul; E-mail: scvrt{at}mahidol.ac.th


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
Human ß-defensin expression correlates with differentiation in oral epithelium, and calcium ion, an important regulator of epithelial differentiation, plays a critical role in regulation of human ß-defensin-2 (hBD-2) mRNA expression. Phospholipase D (PLD) also regulates epithelial differentiation. Therefore, we examined the role of PLD in hBD-2 up-regulation by cell wall extract of Fusobacterium nucleatum and phorbol 12-myristate 13-acetate (PMA), two known hBD-2 activators. We found that hBD-2 mRNA up-regulation in human gingival epithelial cells (HGECs) by these two activators was mediated by PLD activation and blocked by ethanol and 1-butanol, PLD inhibitors. PLD activity was induced by stimulation with these two activators, and phosphatidic acid (PA), a product generated from the PLD enzymatic activity, was detected in stimulated HGECs. Dioctanoyl PA commonly used for PA induced hBD-2 mRNA expression. mRNAs for PLD1{alpha} and ß splice variants as well as PLD1 protein were constitutively expressed, whereas mRNA and protein for PLD2 were expressed at much lower levels than those for PLD1. Moreover, pre-treatment with (±)-propanolol, an inhibitor of phosphatidic acid phosphohydrolases that are the downstream signaling molecules in the PLD pathway, significantly blocked hBD-2 mRNA induction by PMA in a dose-dependent manner. In conclusion, these findings indicate the involvement of PLD activation in hBD-2 up-regulation in HGECs, which correlates with the state of epithelial differentiation.

Keywords: commensal bacteria, gene regulation, gingival epithelial cells, innate immunity, signal transduction


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
One of the functions of epithelia, including the oral epithelium of gingiva, is to produce anti-microbial peptides, such as those of ß-defensin family as part of innate immunity and the epithelial barrier (1). In addition to their anti-microbial activities, the ß-defensins can connect the innate with the acquired immunity by functioning as chemoattractants for immature dendritic cells and T lymphocytes (2) and mast cells (3). We previously reported that human ß-defensin-2 (hBD-2) expression was associated with the state of cellular differentiation (4), consistent with a critical role of Ca2+, an important second messenger in epithelial differentiation (5), in regulating hBD-2 expression in gingival epithelial cells (6). Calcium ion is also an activator of transglutaminases, markers for terminal differentiation that are required for the production of cornified envelopes (7, 8). It was previously demonstrated that sustained phospholipase D (PLD) activation was associated with keratinocyte differentiation (9) and its role in activating transglutaminase activity has recently been demonstrated (10). Therefore, we hypothesized that PLD was a critical component in the signaling pathway for hBD-2, in addition to increased intracellular Ca2+ levels, in response to stimulation with the ubiquitous oral bacterium, Fusobacterium nucleatum, in gingival epithelial cells.

PLD is ubiquitously expressed in animals, plants, fungi and bacteria. It catalyzes the hydrolysis of phospholipids, predominantly phosphatidylcholine at its terminal phosphodiester bond to yield phosphatidic acid (PA) and choline (11). PA, a biologically active molecule that acts as a secondary messenger, can in turn be dephosphorylated by phosphatidic acid phosphohydrolase (PAP) to form diacylglycerol (DAG), which can reversibly be phosphorylated to PA by DAG kinases (12). Similar to the DAG produced by phosphoinositide-specific phospholipase C (PLC), this PLD-generated DAG is thought to activate protein kinase C (PKC). PKC can also be activated directly by phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate, which has been shown to inhibit proliferation and stimulate differentiation of keratinocytes (13). Two related polyphosphoinositol-activated PLD isoforms have been cloned and known as PLD1 (120 kDa) (14) and PLD2 (106 kDa) (15), and PLD1 consists of two splice variants, PLD1{alpha} and PLD1ß (16).

PLD plays a vital role in a variety of biological responses, such as mitogenesis, differentiation, cell signaling, immune responses, inflammation, the processes of endocytosis, secretion and transport (17–19). PLD activity is stimulated by various hormones, neurotransmitters, growth factors, cytokines and other agonists that activate cell-surface G-protein-coupled and tyrosine kinase-linked receptors (20). These stimulants activate PLD activity by multiple pathways, including the Rho family of small G-proteins, ADP-ribosylation factors, Ca2+, PKC{alpha} and protein tyrosine kinases (11, 17, 21). Taken together, since Ca2+ and PKC that can activate PLD are critical for hBD-2 regulation (6, 22), it was therefore likely that PLD was another important signaling molecule in hBD-2 up-regulation.

In this report, we examined the role of PLD in hBD-2 up-regulation by cell wall extract of F. nucleatum, a Gram-negative periodontal bacterium known to be an hBD-2 activator (23). We found that hBD-2 up-regulation by F. nucleatum cell wall extract was mediated by activation of PLD enzymes. Consistent with this finding, the short-chain L-{alpha}-phosphatidic acid, dioctanoyl C8:0 (DOPA) could induce hBD-2 mRNA expression. We also examined mRNA and protein expression for PLD1{alpha} and PLD1ß splice variants and PLD2 as well as the presence of PA mass and the PLD enzymatic activity in stimulated human gingival epithelial cells (HGECs). Our results demonstrate the involvement of PLD activation in regulating hBD-2 expression in addition to its role in activating transglutaminase activity as previously reported (10).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Materials
DOPA, (±)-propanolol and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma–Aldrich, St Louis, MO, USA. All chemical reagents were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO was <0.1% (v/v of culture medium). The final concentration of PMA was100 ng ml–1; those of DOPA were 25, 50 and 100 µg ml–1 and those of (±)-propanolol were 10, 30 and 100 µM. Ethanol, 1-butanol and tertiary butanol (t-butanol) were of analytical grade with >99% in purity. The cell wall extract of F. nucleatum was prepared as described previously (24). The mAb against the N-terminal residues (F-12) of human PLD1 and the polyclonal antibody against human PLD2 (N-20) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA. The polyclonal antibody against the phosphorylated form of PLD1 at threonine 147 was from Cell Signaling Technology, Beverly, MA, USA.

Cell culture
HGECs were isolated from gingival tissue overlying impacted third molars as described previously (24) and collected in accordance with approved Human Subject Policies. HGECs were cultured in serum-free keratinocyte growth medium (KGM) (BioWhittaker Inc., Walkersville, MD, USA), containing low calcium (0.03 mM). After 80% confluence, HGECs were stimulated with either 3, 10 and 30 µg ml–1 of F. nucleatum cell wall extract or 1, 10 and 100 ng ml–1 of PMA for indicated times in the absence or presence of various doses of (±)-propanolol, ethanol, 1-butanol and t-butanol. In addition, HGECs were stimulated with 25, 50 and 100 µg ml–1 of DOPA.

Isolation of total RNA and reverse transcription–PCR
Total RNA was isolated by the Aurum Total RNA Mini Kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instruction. Samples of total RNA were quantified by optical density reading at 260 nm. The mRNAs for hBD-2, PLD1{alpha} and ß splice variants, PLD2 and transglutaminase 1 (TGM1) were detected by means of reverse transcription (RT)–PCR. Briefly, 3 µg of total RNA sample was used for the synthesis of cDNA by the SuperScriptTM First-Strand cDNA Synthesis System for RT–PCR (Fermentas, Hanover, MD, USA). The RT–PCR protocol was previously described (23), and PCR was performed in a Mastercycler Gradient thermal cycler (Eppendorf, Germany) for various cycles (28 cycles for hBD-2 and TGM1, 20 cycles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 30 cycles for PLD1 and 35 cycles for PLD2). PCR primers for amplification of hBD-2 and GAPDH were as previously used (6). The primer sequences and the PCR conditions for PLD1{alpha} and ß splice variants and PLD2 as well as those for TGM1 were as previously described (25, 26, respectively). The PCR products were resolved on a 1.2% agarose gel in 1x Tris borate EDTA (TBE) and visualized with ethidium bromide (Bio-Rad Laboratories) staining. Photographs of gels were taken by a CCD camera attached to the Gel Documentation 1000 (Bio-Rad Laboratories). The identities of the amplified products for the PLD1{alpha} and ß splice variants, PLD2 isoform and TGM1 were verified by DNA sequencing at the Sequencing Facility, Medical Science Research Equipment Center, Faculty of Medicine, Chiang Mai University, Thailand. The size of PCR product for TGM1 (264 bp) was as predicted, and the sequence comparison of PCR product for TGM1 resulted in 100% identity to the gene for human TGM1 enzyme (accession number M62925). The size of PCR products for PLD1{alpha} (446 bp) and ß (332 bp) splice variants and PLD2 isoform (329 bp) was as predicted (25), and the nucleotide sequence of PCR product was identical to the published human sequence for PLD1 and PLD2 (16, 27, respectively). The mean ratios of two PLD1 splice variants or PLD2 relative to GAPDH from three separate experiments were determined by the densitometry of PCR products using the Gel Documentation 1000 equipped with Molecular Analyst software version 1.4 and plotted on a bar graph in Fig. 3(E).


Figure 3
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  		Fig. 3. Expression of PLD1 and PLD2 mRNA and protein and their activity in HGECs. (A) RT–PCR analysis. HGECs were stimulated with various doses of Fusobacterium nucleatum cell wall extract or PMA overnight or left untreated as a control. Total RNA isolation and RT–PCR was conducted as described in Methods. (B) Western blot analysis. HGECs were stimulated with F. nucleatum cell wall extract or PMA for indicated times (0–24 h). Total protein extraction and western blot analysis using primary antibodies against the non-phosphorylated and the phosphorylated form of PLD1 or PLD2 were conducted as described in Methods. The data shown in (A) and (B) were representative of three separate experiments. (C) Assay for PLD activity in HGECs. HGECs were stimulated with 10 µg ml–1 of F. nucleatum cell wall extract or 100 ng ml–1 of PMA, and the assay for PLD activity was conducted as described in Methods. Data were given as mean PLD activity in mU ml–1 ± SD. of three independent experiments. (D) Thin-layer chromatogram showing time course of PA formation in HGECs (5 x 106 cells) upon incubation with F. nucleatum cell wall extract for various times, and the stimulation was stopped with lipid extraction. Lipid extract and 0.12 mM of dioleoyl PA were separated by TLC as described in Methods. (E) Densitometry of mRNA expression for two PLD1 splice variants and PLD2 in control, F. nucleatum-stimulated (+F.n.CW) and PMA-stimulated (+PMA) samples. The y-axis of a bar graph represented the mean ratios of mRNA expression for the {alpha} (filled bars) and ß (empty bars) splice variants of PLD1 and PLD2 (stippled bars) normalized by the levels of GAPDH expression of three separate experiments. The lowest ratio of PLD2 expression relative to GAPDH was set at one.

 
Preparation of plasmid vectors for hBD-2, TGM1 and GAPDH
Inserts for GAPDH, hBD-2 and TGM1 were prepared by PCR of cDNA using a specific primer pair for GAPDH (forward primer, 5'-CATCCATGACAACTTTGGTA-3' and reverse primer, 5'-TTACTCCTTGGAGGCCATGT-3'), hBD-2 (forward primer, 5'-GGTGAAGCTCCCAGCCATCAG-3' and reverse primer, 5'-CATCTTTGGACACCATAGTTT-3') and TGM1 (forward primer, 5'-GCATCTGTGGGTCCTGTCCCATCCAT-3' and reverse primer, 5'-CTCGACCCCTGGAGTCAGAGGGTTCAG-3'). The plasmid vectors were prepared as described previously (28). The concentration of each plasmid vector was determined by UV absorbance and diluted to nanogram levels to be used as standards for real-time PCR.

Real-time PCR
The real-time PCR experiment was performed in triplicate using 10% (v/v) of cDNA, prepared from 3 µg of total RNA and the Light-Cycler-FastStart DNA Master SYBR Green I system (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer's protocol. 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–1. The fluorescence activity was measured at 530 nm during the extension phase. The levels of mRNA expression for hBD-2, TGM1 and GAPDH in each sample were determined by comparing the fluorescence intensity in each sample during the increasing phase of amplification with the intensity of the known concentrations of standard hBD-2, TGM1 and GAPDH plasmid. The mean relative ratios of both hBD-2 to GAPDH and TGM1 to GAPDH in each control or inhibitor-treated samples were calculated and compared with those in the F. nucleatum- or PMA-stimulated sample set at 100%.

Western blot analysis
HGECs at 80% confluence were stimulated with 10 µg ml–1 of F. nucleatum cell wall extract or 100 ng ml–1 of PMA for various times. Whole-cell lysates were extracted in RIPA buffer (28). The lysates were then transferred to 1.5-ml microcentrifuge tubes, vortexed vigorously and centrifuged briefly. Protein content in the supernatant was determined by protein assay (Bio-Rad Laboratories) with {gamma}-globulin as a standard. Forty micrograms of cell lysates were resolved on a 7.5% SDS–PAGE and were then transferred to nitrocellulose membranes (Bio-Rad Laboratories) for 12 h in the cold. Biotinylated protein markers (Cell Signaling Technology) were run along with the samples. The membranes were blocked and incubated with primary antibodies against the phosphorylated and the non-phosphorylated form of PLD1 or PLD2 diluted in appropriate blocking buffer. The membranes were then incubated with HRP-conjugated secondary antibody (KPL, Gaithersburg, MD, USA) at 1/2000 in 10 ml of blocking buffer for 1 h at room temperature with gentle agitation and were then incubated with the LumiGLO Reserve Chemiluminescent substrate (KPL). The excess developing solution was drained off, and the membranes were subsequently exposed to HyperfilmTM ECL (Amersham Biosciences, Buckinghamshire, UK).

Assay for PLD activity
After HGECs were stimulated with 10 µg ml–1 of F. nucleatum cell wall extract or 100 ng ml–1 of PMA for various times, KGM was replaced with 300 µl of ice-cold 50 mM Tris–HCl, pH 8.0. HGECs were broken by three freeze and thaw cycles. Samples were collected, and 100 µl of the samples were mixed with 100 µl of the Amplex Red reaction buffer (Amplex® Red Phospholipase D assay kit, Molecular Probes, Inc., Eugene, OR, USA). The PLD activity was assayed in triplicate for each sample by determining the fluorescence activity after 1-h incubation at 37°C in the dark with the SynergyTM HT Multi-Detection Microplate Reader (BioTek Instruments, Inc., Winooski, VA, USA). A standard curve was performed with purified PLD from Streptomyces chromofuscus (Sigma–Aldrich, catalog number P8023), whose concentrations ranged from 0 to 500 mU ml–1. The experiment was repeated three times using different cell lines derived from different donors, and similar results were obtained.

Extraction and thin-layer chromatography of lipids
Total lipids from HGECs were extracted according to the method of Bligh and Dyer (29). PA was separated from other lipids by one-dimensional thin-layer chromatography (TLC) on silica gel 60 F254 aluminum-backed thin-layer plates. Dioleoyl PA from Sigma–Aldrich (catalog number P2767) was separated along with the lipid samples as a PA standard. PA was resolved from other lipids by a three-step one-dimensional TLC modified from Tou et al. (30). Briefly, the chromatogram was first developed in a solvent system consisting of ethyl acetate/acetic acid/2,2,4-trimethylpentane (9:2:5, by volume). After the chromatogram had been hood-dried for 1 h, it was cut 1 cm above the PA standard to remove the neutral lipids. It was turned 180° and developed in chloroform/methanol/28% ammonium/water (40:20:3:1, by volume) until the solvent moved to the sample origin on the chromatogram. The chromatogram had been hood-dried for 1 h, and it was turned 180° and developed in the second solvent system. After the chromatogram had been hood-dried for 1 h, it was stained with iodine vapor for 5 min and the chromatogram pictures were scanned by an HP LaserJet 3020.

Statistical analysis
The differences in terms of the percentage of hBD-2 induction between the stimulated sample and the inhibitor-treated samples in Figs. 2(B) and 4(C) were tested by One-way analysis of variance at the significant level P < 0.05 or P < 0.01.


Figure 2
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  		Fig. 2. Involvement of PLD in hBD-2 mRNA induction. (A) RT–PCR analysis. HGECs were pre-treated with indicated percentages (v/v) of ethanol, 1-butanol or t-butanol for 30 min prior to stimulation with Fusobacterium nucleatum cell wall extract overnight. (B) Real-time PCR assay. The y-axis represented the mean percentage of hBD-2 (empty bars) or TGM1 (stippled bars) mRNA induction in the control and inhibitor-treated samples in comparison to the F. nucleatum-stimulated sample, which was set at 100%. The results were represented as means plus standard deviations of four separate experiments. HBD-2 mRNA induction was significantly inhibited by 2% of ethanol, 0.6 and 1% of 1-butanol (**P < 0.01).

 

Figure 4
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  		Fig. 4. Involvement of PA and PAP in hBD-2 mRNA induction. (A) RT–PCR analysis. HGECs were stimulated with indicated doses of DOPA or Fusobacterium nucleatum cell wall extract as a positive control. (B) RT–PCR analysis. HGECs were pre-treated with indicated doses of (±)-propanolol for 30 min prior to stimulation with either F. nucleatum cell wall extract or PMA overnight. The results shown in (A) and (B) were representative of four separate experiments. (C) Real-time PCR assay. HBD-2 mRNA induction by PMA was significantly inhibited by 10 µM of (±)-propanolol (*P < 0.05) and by 30 and 100 µM of (±)-propanolol (**P < 0.01). The results were shown as means plus standard deviations of four separate experiments in (B).

 

   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Induction of TGM1 and hBD-2 mRNA by F. nucleatum cell wall extract and PMA
We previously reported that expression of human ß-defensins correlated with the state of oral epithelial differentiation (4). Consequently, to determine whether stimulation with F. nucleatum cell wall extract would induce differentiation in HGECs, we investigated mRNA expression for TGM1, a marker for terminal differentiation. Both hBD-2 and TGM1 mRNAs were up-regulated by F. nucleatum cell wall extract as well as by PMA, a known activator for transglutaminase activity (9) (Fig. 1A). Interestingly, PMA appeared to induce TGM1 mRNA expression greater than F. nucleatum cell wall extract did, indicating different natures between two distinct stimulants, i.e. while PMA is well known for its effect on keratinocyte differentiation (13), F. nucleatum tends to induce host immune responses rather than differentiation. The time-course study showed that TGM1 mRNA was transiently induced after HGECs were stimulated with F. nucleatum cell wall extract and PMA for 6 h (Fig. 1B). Consistent with our previous study (23), early hBD-2 mRNA induction by F. nucleatum cell wall extract (3 h) and late hBD-2 induction by PMA (12 h) were demonstrated (Fig. 1B). It is probable that the different kinetic profiles between hBD-2 and TGM1 mRNA expression by two different stimulants may reflect the complex interactions and influences from other signaling molecules that are possibly involved, although PLD was subsequently shown as one of the important signaling molecules in controlling these two genes.


Figure 1
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  		Fig. 1. Induction of TGM1 and hBD-2 mRNA by Fusobacterium nucleatum cell wall extract and PMA. (A) RT–PCR analysis. HGECs were stimulated with indicated doses of F. nucleatum cell wall (F.n.CW) extract or PMA overnight or left untreated as a control. Total RNA isolation and RT–PCR was conducted as described in Methods. (B) RT–PCR analysis. HGECs were stimulated with F. nucleatum cell wall extract or PMA for indicated times (0–48 h). The time-course study demonstrated transient up-regulation of TGM1 mRNA from 6 to 24 h. The results shown were representative of three separate experiments.

 
Involvement of PLD activation in hBD-2 mRNA induction
Since TGM1 was up-regulated by F. nucleatum cell wall extract and PMA (Fig. 1A) and the transglutaminase activity was previously shown to be activated via PLD signaling (10), we asked whether hBD-2 up-regulation was also mediated by PLD activation. In the presence of ethanol and 1-butanol, the PLD catalyzes transphosphatidylation reaction that generates phosphatidylethanol and phosphatidylbutanol, respectively (31). This reaction is very specific for primary short-chain alcohols as tertiary alcohols, like t-butanol, are not acceptors of the phosphatidyl group. Therefore, t-butanol is commonly used as a negative control for the inhibitory effect on PLD by 1-butanol (32, 33). Unlike PA, these phosphatidylalcohols are not normal constituents of cells or biological membranes and cannot mediate the PLD-signaling pathway. Pre-treatment with ethanol and 1-butanol either partially or completely inhibited hBD-2 mRNA induction by F. nucleatum cell wall extract (Fig. 2A), and the real-time PCR analysis showed statistically significant inhibition by 2% (v/v) of ethanol and 0.6 and 1% (v/v) of 1-butanol (P < 0.01; Fig. 2B). Pre-treatment with 1% (v/v) of t-butanol did not inhibit hBD-2 mRNA induction by F. nucleatum cell wall extract (Fig. 2A), suggesting a specific inhibition by primary alcohols and involvement of PLD. Consistent with the inhibition of hBD-2 mRNA induction by primary alcohols, TGM1 mRNA induction was blocked by ethanol and 1-butanol, but not t-butanol (Fig. 2A). The real-time PCR analyses showed a complete inhibition of TGM1 mRNA induction by 2% (v/v) of ethanol and 0.6 and 1% (v/v) of 1-butanol (Fig. 2B). The potential for non-specific effects due to cell toxicity from primary alcohols was not found in this study since total RNA yield from alcohol-treated samples did not differ from that of control untreated sample. Moreover, relatively equal amount of GAPDH mRNA expression was demonstrated between samples (Fig. 2A). The concentrations of alcohols used in this study, i.e. up to 1% (v/v) of 1-butanol and 2% (v/v) of ethanol, were previously used to block cyclooxygenase-2 (COX-2) expression (34) and to decrease sulfur mustard-induced arachidonic acid release (35).

Expression of PLD1 and PLD2 mRNA and protein and their activity in HGECs
In HGECs, PLD1{alpha} and ß splice variants were constitutively expressed, while PLD2 mRNA was up-regulated by all doses of PMA tested (Fig. 3A). The intensity of the PLD1{alpha} band was less than that of PLD1ß (Fig. 3A and E), suggesting that HGECs preferentially express the latter splice variant. According to the densitometry, both PLD1{alpha} and ß splice variants were expressed in much greater levels than was PLD2 by ~10- and 37-fold in control and F. nucleatum-stimulated samples and by 9- and 27-fold in PMA-stimulated sample, respectively (Fig. 3E). Consistently, expression for PLD2 protein at 106 kDa was detected at the low level in HGECs (Fig. 3B). Consistent with constitutive mRNA expression for two PLD1 splice variants, two immunoreactive bands at 120 and 80 kDa were detected with the PLD1 antibody (an arrow and an arrowhead, respectively, Fig. 3B), and there was no alteration in PLD1 protein expression in response to both stimulants. Transient activation of the PLD1 by phosphorylation from 30 min to 6 h was observed in PMA-stimulated, but not F. nucleatum-stimulated, HGEC lysates (Fig. 3B). This was consistent with the findings from a previous study (36) that demonstrated PLD1 activation in vivo by phosphorylation occurring at multiple sites, including threonine 147, in response to PMA-induced PKC activation. Collectively, these results suggest the mechanism of PLD1 activation by phosphorylation following PMA stimulation of the cells, while that of PLD1 activation in response to F. nucleatum cell wall extract stimulation is unknown and subject to further investigation. The functional activity of PLD enzymes in HGECs was monitored indirectly using 10-acetyl-3,7-dihydrophenoxazine (Amplex Red reagent), a sensitive fluorogenic probe for H2O2. The PLD activity was transiently induced when HGECs were stimulated with F. nucleatum cell wall extract from 3 to 12 h (Fig. 3C), corresponding with an early hBD-2 induction by F. nucleatum cell wall extract (23; Fig. 1B). It was interesting to note that the induction of PLD activity by F. nucleatum cell wall extract was significantly reduced at 24 h, whereas the level of hBD-2 mRNA expression at 24 h was still high. It is possible that PLD enzymes function as intermediate molecules and other downstream signaling molecules in the PLD pathway, such as PA, may participate in hBD-2 up-regulation and/or hBD-2 mRNA is stable and accumulated during the prolonged stimulation. In addition, the PLD activity was markedly induced after being stimulated with PMA for 12 h (Fig. 3C), consistent with late hBD-2 induction by PMA (23; Fig. 1B). Consistent with the induced PLD activity, PA mass was elevated in the lipid extracts from HGECs stimulated with F. nucleatum cell wall extract from 3 to 24 h (Fig. 3D). In contrast, the changes in PA mass were not detected with the present method in control HGECs (Fig. 3D). Interestingly, PA mass was still detected at 24 h (Fig. 3D), although the PLD activity was significantly reduced (Fig. 3C). This may be because DAG kinases can generate PA from DAG (12) in addition to PLD enzymes that generate PA from phosphatidylcholine.

Involvement of PA and PAP in hBD-2 mRNA induction
Although the experiments in Fig. 2(A) showed that PLD was involved in hBD-2 up-regulation by F. nucleatum cell wall extract, they provided no direct evidence that PA was involved in hBD-2 up-regulation. To test this, HGECs were treated with various concentrations of DOPA, which is commonly used for PA to see the direct effect of PA (34, 37). It was found that hBD-2 mRNA was induced by DOPA (Fig. 4A), confirming an important role of PLD enzymes and their derived product, i.e. PA, in hBD-2 up-regulation. It was interesting to note that the amount of PA generated from stimulating HGECs with 10 µg ml–1 of F. nucleatum cell wall extract was approximately equivalent to 80 µg ml–1 or 0.12 mM of standard dioleoyl PA (Fig. 3D). This was consistent with the greater hBD-2 mRNA induction by 10 µg ml–1 of F. nucleatum cell wall extract than by 25 µg ml–1 of DOPA (Fig. 4A). To further establish the downstream signaling pathway of hBD-2 up-regulation, we treated HGECs with various doses of (±)-propanolol, which is a PAP inhibitor (35), prior to stimulation. HBD-2 mRNA induction by PMA, but not by F. nucleatum cell wall extract, was inhibited by pre-treatment with (±)-propanolol in a dose-dependent fashion (Fig. 4B). Particularly, 100 µM of (±)-propanolol significantly inhibited hBD-2 mRNA induction by PMA almost 50% (P < 0.01) (Fig. 4C). This suggests that PAPs are downstream signaling molecules for PLD in hBD-2 up-regulation by PMA. However, other enzymes than PAPs, like phospholipases A1/A2 that generate another bioactive lipid, i.e. lysophosphatidic acid (38), rather than DAG, may be responsible for hBD-2 up-regulation by F. nucleatum cell wall extract in HGECs.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
In this study, we report several new findings on regulation of the anti-microbial peptide hBD-2 in epithelial cells. First, our study shows that activation of PLD is important for hBD-2 mRNA induction in HGECs in response to the commensal periodontal bacterium, F. nucleatum. In addition to its role in hBD-2 regulation, PLD is involved in up-regulation of TGM 1, a marker for terminal differentiation, consistent with our previous study and others regarding the association between hBD-2 expression and keratinocyte differentiation (4, 39). The inhibitory effect on PLD by primary alcohol like 1-butanol is specific since tertiary alcohol, t-butanol, whose chemical formula is identical to 1-butanol, does not block hBD-2 mRNA up-regulation (Fig. 2A and B). Nevertheless, it should be cautious when the results from the study using pharmacological inhibitors are interpreted possibly due to their non-specific inhibition. Consequently, another approach, i.e. transfection with RNA interference, is required to corroborate the involvement of a specific PLD isoform in hBD-2 up-regulation. Second, we show the constitutive mRNA expression for PLD1{alpha} and ß splice variants and the inducible mRNA expression for PLD2 isoform by PMA in HGECs. To the best of our knowledge, there has been no previous study that showed mRNA and protein expression for PLD1 and PLD2 in primary gingival epithelial cells. We show that the PLD1ß splice variant is preferentially expressed in HGECs, consistent with its expression in the human keratinocyte cell line HaCaT (40); however, expression of the PLD1{alpha} splice variant is seen only in HGECs, reflecting a difference between primary cells and an immortalized cell line.

Third, the full length form of PLD1 at 120 kDa as well as its possible fragment at 80 kDa are detected in HGEC whole-cell lysates, and this lower band may be comparable with the presence of the lower bands below 120 kDa detected in the HaCaT lysates (40). In contrast to transient PLD1 phosphorylation by PMA, PLD1 phosphorylation in response to stimulation with F. nucleatum cell wall extract is not detected (Fig. 3B). In fact, there have been several studies showing the PLD activation in the absence of ATP, indicating a non-phosphorylation mechanism (41–43). Therefore, it is possible that the PLD1 is not directly phosphorylated by stimulation with F. nucleatum cell wall extract in vivo, but may be activated by other mechanisms instead in order to regulate signaling events. The induction of PLD activity at 3 h by F. nucleatum cell wall extract (Fig. 3C) is in agreement with the detectable PA mass at 3 h (Fig. 3D) and the early hBD-2 mRNA induction (Fig. 1B). This confirms the importance of PLD activation in hBD-2 mRNA up-regulation by F. nucleatum cell wall extract. Last, the importance of PLD in hBD-2 regulation is corroborated by the fact that the short-chain DOPA can itself induce hBD-2 mRNA expression (Fig. 4A) regardless of stimulation with F. nucleatum cell wall extract.

The roles of several agonists in PLD activation have been demonstrated in many studies, and many of these act through G-protein-coupled or growth factor receptors. Most of the agonists that trigger PLD also stimulate phosphatidylinositol-4,5-bisphosphonate-specific PLC. It has been demonstrated that PLC activation results in an increase in intracellular Ca2+ and PKC activation that can subsequently activate PLD (11). Our study and others previously demonstrated the role of Ca2+ and PKC in hBD-2 mRNA up-regulation (6, 22). Therefore, it is probable that PLC is activated rapidly in response to agonists and plays an initial role, whereas PLD, which is activated by an increase in intracellular Ca2+ and PKC activation that result from PLC activation, plays a subsequent role in hBD-2 regulation, but a more sustained cellular response as long as the stimulants are present. In addition to Ca2+ and PKC, activation of p38 mitogen-activated protein (MAP) kinase has been shown to induce PLD activity (44). We have shown that hBD-2 mRNA up-regulation by F. nucleatum cell wall extract in HGECs is via phosphorylation of both p38 and c-jun N-terminal kinase (JNK) MAP kinases (28). Consequently, it is interesting to further delineate multiple signaling pathways as well as the roles of these interconnecting signaling molecules, including PLC, PLD, PKC, Ca2+, p38 and JNK MAP kinases, in regulation of hBD-2 expression.

Propanolol, a PAP inhibitor, has been frequently used to distinguish the relative contributions of lysophosphatidic acid versus DAG in a given physiological response. In mammalian cells, conversion of PA to lysophosphatidic acid is catalyzed by phospholipase A1/A2 activity (45), while the formation of DAG from PA is regulated by PAP (46). Since hBD-2 mRNA induction by PMA, but not F. nucleatum cell wall extract, is inhibited by propanolol, it is therefore interesting to further investigate different downstream signaling molecules mediated by these two stimulants despite the fact that PLD enzymes play an important role in hBD-2 mRNA induction.

With respect to the physiological relevance, PLD enzymes play an essential role in the regulation of keratinocyte differentiation (47). It was previously shown that 1,25-dihydroxyvitamin D3, a sterol hormone known to induce differentiation, specifically increased the expression and activity of PLD1, but not PLD2 (48). mRNA expression for PLD1 was observed in the spinous and granular layers, with little or no expression in the basal layer (48), which was consistent with the localization of hBD-2 mRNA expression in gingival epithelium (4). Moreover, it has recently been shown that 1,25-dihydroxyvitamin D3 is a direct inducer of hBD-2 (49), and up-regulated expression of the vitamin D-related genes, including vitamin D receptor and vitamin D-1-hydroxylase genes, leads to induction of another human anti-microbial peptide, cathelicidin, and its active peptide LL-37 via activation of Toll-like receptor 2/1 (50). Together, all these findings support a link between vitamin D-mediated innate immune responses by up-regulation of anti-microbial peptide expression and keratinocyte differentiation via PLD activation. It has also been shown that PLD enzymes play an important role in activation of expression and secretion of other effector molecules in innate immune responses, i.e. IL-8 and Cox-2, in simple epithelial tissues (32, 33, 51), in addition to their role in up-regulation of anti-microbial peptide hBD-2 and keratinocyte differentiation. In summary, the findings from this study demonstrate the importance of PLD activation in hBD-2 up-regulation in stratified oral epithelia, which correlates well with the state of cellular differentiation. The role of PLD in regulating effector molecules in innate immune responses, particularly anti-microbial peptides, has not been previously recognized and may have implications for potential therapeutic applications in up-regulation of these natural anti-microbial peptides.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Thailand Research Fund (RSA 4780003 to S. K.); Senior Research Scholar Award to V. R.; the Post-graduate Education and Research Program in Chemistry and the National Research Council of Thailand to P.K.


   Acknowledgements
 
We would like to acknowledge Beverly A. Dale for careful reading and helpful suggestions for the manuscript. We thank Tongkam Taya and Sineenart Santidherakul at the Medical Science Research Equipment Center, Faculty of Medicine, Chiang Mai University, for their technical assistance in the sequencing reaction and the assay for PLD activity, respectively.


   Abbreviations
 
COX-2, cyclooxygenase-2
DAG, diacylglycerol
DOPA, L-{alpha}-phosphatidic acid, dioctanoyl C8:0
DMSO, dimethyl sulfoxide
GAPDH, glyceraldehyde-3-phosphate dehydrogenase
hBD-2, human ß-defensin-2
HGEC, human gingival epithelial cell
JNK, c-jun N-terminal kinase
KGM, keratinocyte growth medium
MAP, mitogen-activated protein
PA, phosphatidic acid
PAP, phosphatidic acid phosphohydrolase
PKC, protein kinase C
PLC, phospholipase C
PLD, phospholipase D
PMA, phorbol 12-myristate 13-acetate
RT, reverse transcription
t-butanol, tertiary butanol
TGM1, transglutaminase 1
TLC, thin-layer chromatography

   Notes
 
Transmitting editor: S. H. E. Haufman

Received 2 February 2007, accepted 11 October 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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