JDR JDR Most Cited Articles
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
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 ISI Web of Science (12)
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.
J Dent Res 82(11): 877-882, 2003
© 2003 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Intracellular Calcium in Signaling Human ß-Defensin-2 Expression in Oral Epithelial Cells

S. Krisanaprakornkit1,*, D. Jotikasthira2, and B.A. Dale3

1 Department of Odontology-Oral Pathology,
2 Department of Orthodontics, Faculty of Dentistry, Chiang Mai University, Chiang Mai 50200, Thailand; and
3 Department of Oral Biology, School of Dentistry, University of Washington, Seattle, WA 98195, USA;

* corresponding author, sutichai{at}chiangmai.ac.th, suttichaikris{at}yahoo.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of human ß-defensins is correlated with differentiation in the oral epithelium, consistent with their function as part of the epithelial antimicrobial barrier. Because calcium is a known regulator of epithelial differentiation, we tested the hypothesis that calcium concentration mediates ß-defensin expression. Gingival epithelial cells were cultured in medium containing low calcium concentration (0.03 mM), then either changed to high extracellular calcium concentrations or stimulated with thapsigargin to release intracellular calcium stores in the presence or absence of BAPTA-AM, a calcium chelator. Human ß-defensin-2 (hBD-2) mRNA expression was rapidly induced by thapsigargin, and more slowly induced by high extracellular calcium. Induction of hBD-2 peptide was confirmed by immunofluorescence. BAPTA-AM inhibited hBD-2 induction by both thapsigargin and calcium in a dose-dependent fashion. In addition, BAPTA-AM inhibited hBD-2 induction by a bacterial stimulant. Collectively, these findings demonstrate that intracellular calcium is a critical mediator of hBD-2 expression. Abbreviations used in this study are: BAPTA-AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid tetrakis (acetoxymethyl ester); DMSO, dimethylsulfoxide; F. nucleatum, Fusobacterium nucleatum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBDs, human ß-defensins; HGECs, human gingival epithelial cells; MAP, mitogen-activated protein; and RT-PCR, reverse-transcriptase/polymerase chain-reaction.

KEY WORDS: innate immunity • antimicrobial peptide • ß-defensins • calcium • thapsigargin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oral stratified epithelia function as a mechanical barrier, but in addition, they function as an active antimicrobial barrier via the production of antimicrobial peptides, such as those of the ß-defensin family (reviewed in Dale, 2002). The ß-defensins are a component of the innate immune system and also bridge innate and acquired immune responses by acting as chemoattractants for mast cells (Niyonsaba et al., 2002), immature dendritic cells, and T-lymphocytes (Yang et al., 1999). Several ß-defensins are found in oral epithelia. Human ß-defensin-1 is widely distributed in epithelia (Zhao et al., 1996), including oral epithelial cells and tissue. HBD-2, originally identified in psoriatic skin (Harder et al., 1997), is also expressed in oral epithelia (Krisanaprakornkit et al., 2000). Expression of hBD-1 in oral epithelial cells is constitutive (Krisanaprakornkit et al., 1998; Mathews et al., 1999), while hBD-2 is up-regulated in response to bacterial stimulants, phorbol ester, TNF-{alpha} (Krisanaprakornkit et al., 2000), and IL-1 (Mathews et al., 1999). HBD-1 and -2 demonstrate a broad spectrum of antimicrobial activity against both Gram-negative and Gram-positive bacteria in vitro (Valore et al., 1998). Another ß-defensin, hBD-3, more specific for Gram-positive organisms, is induced by TNF-{alpha} and bacteria in cultured keratinocytes and airway epithelial cells and is also found in oral tissues (Harder et al., 2001; Dunsche et al., 2002).

Several studies suggest the association between hBD-1 and -2 expression and differentiation (Dale et al., 2001; Liu et al., 2002). In situ hybridization revealed the localization of hBD-1 and hBD-2 mRNA within the suprabasal keratinocytes (Fulton et al., 1997; Dale et al., 2001). In cultured skin keratinocytes, hBD-2 induction is seen in large differentiating cells (Liu et al., 2002). Tissue immunolocalization shows hBD-1 and hBD-2 peptides within the upper portion of the epithelium, consistent with their role in the antimicrobial barrier (Ali et al., 2001; Dale et al., 2001). Calcium is an important regulator of epithelial differentiation. Keratinocytes grown in low calcium concentration are proliferative, but when shifted to media containing higher calcium concentrations, they stratify and express differentiation markers such as involucrin, K1, K10, loricrin, and profilaggrin (Yuspa et al., 1989). Calcium also stimulates the activity of transglutaminases, necessary for the production of cornified envelopes (Polakowska and Goldsmith, 1991; Missero et al., 1996). The role of calcium in differentiation in vivo is reflected in the presence of a calcium gradient in epidermis that increases from the basal to the granular cell layer (Menon and Elias, 1991). Because expression of hBD-1 and -2 is associated with or regulated by the state of differentiation in oral epithelia and epidermis (Dale et al., 2001; Liu et al., 2002), we postulated that calcium is an important component of molecular signaling for the expression of human ß-defensins.

In this study, we show that hBD-2 mRNA and peptide regulation is mediated by extracellular and intracellular calcium. We find that the kinetics of hBD-2 mRNA induction by high extracellular calcium is slower than that in cells stimulated with thapsigargin, which increases intracellular calcium. Moreover, we demonstrate the calcium-dependent hBD-2 up-regulation by cell wall extract of F. nucleatum, an oral commensal bacterium. This work has implications for the molecular mechanisms involved in the expression of antimicrobial peptides in relation to epithelial differentiation.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
HGECs were isolated from gingival tissue overlying impacted third molars as described previously and collected in accordance with approved Human Subject policies (Krisanaprakornkit et al., 1998). HGECs were cultured in serum-free keratinocyte growth medium (BioWhittaker Inc., Walkersville, MD, USA), containing low calcium (0.03 mM). After 80% confluence, HGECs were stimulated with either 10 µg of F. nucleatum cell wall extract/mL, calcium (0.03, 0.06, 0.15, 0.60, and 1.20 mM), or thapsigargin (Sigma, St. Louis, MO, USA) for 1, 3, 6, 12, 18, or 24 hrs in the presence or absence of BAPTA-AM (Sigma). Thapsigargin and BAPTA-AM were dissolved in DMSO and added to the culture medium so that the concentration was less than 0.1% of the total volume. The final concentrations of thapsigargin were 1, 10, 100, and 1000 nM; those of BAPTA-AM were 10 and 30 µM. The cell wall extract of F. nucleatum was prepared as described previously (Krisanaprakornkit et al., 1998).

Isolation of Total RNA and RT-PCR
Total RNA was isolated by TRIZOL® LS reagent (Life Technologies, Inc., Rockville, MD, USA) according to the manufacturer’s instructions. Samples of total RNA were quantified by optical density reading at 260 nm. The ß-defensin mRNAs were detected by means of RT-PCR. Briefly, a 3-µg quantity of each total RNA sample was used for the synthesis of cDNA by the SuperScriptTM First-Strand Synthesis System for RT-PCR (Invitrogen, Life Technologies, Inc.). The RT-PCR protocol was previously described (Krisanaprakornkit et al., 2000), and PCR was performed in a Mastercycler Gradient thermal cycler (Eppendorf, Germany) for 28 cycles, according to the following steps: (1) 30 sec at 95°C; (2) 30 sec at 60 or 65°C; and (3) 1 min at 72°C. PCR primers for amplification of hBD-1, hBD-2, IL-8, and GAPDH were as previously used (Krisanaprakornkit et al., 1998, 2000; Dale et al., 2001). The primers for hBD-3 were 5'-TGA AGC CTA GCA GCT ATG AGG-3' (forward) and 5'-AGC ACT TGC CGA TCT GTT CCT-3' (reverse). The PCR products were resolved on a 1.5% agarose gel in 1X TBE and visualized with ethidium bromide staining. Photographs of gels were taken by a CCD camera attached to the Gel Documentation 1000 (Bio-Rad Laboratories, Hercules, CA, USA) equipped with Molecular Analyst software version 1.4 for analysis of the densities of PCR products. The ratio between hBD-2 and GAPDH mRNA expression in each experimental sample and control was calculated and plotted on a bar graph. Each experiment was performed independently at least three times with cell lines derived from different donors, and the ratios were shown by means ± SD. The identity of the amplified products for hBD-3 was verified by DNA sequencing at the Sequencing Facility, Medical Science Research Equipment Center, Faculty of Medicine, Chiang Mai University, Thailand. The identities of the amplified products for hBD-1 and hBD-2 were previously characterized (Krisanaprakornkit et al., 1998, 2000, respectively).

Immunolocalization of hBD-2
To detect the hBD-2 peptide, we grew HGECs on coverslips and incubated them with either 1.20 mM calcium, 1000 nM thapsigargin, or cell wall extract of F. nucleatum (a control for an hBD-2 activator), or left them unstimulated overnight. HGECs were fixed in 4% paraformaldehyde in Sorensen’s buffer and permeabilized with cold acetone on ice for 5 min. For immunostaining, cells were blocked with 3% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 20 min, and then incubated with polyclonal antibody against hBD-2 (1:500) or pre-immune rabbit serum, a generous gift from Dr. Tomas Ganz, Department of Medicine, UCLA, USA. Cells were rinsed, reacted with FITC-conjugated secondary goat anti-rabbit IgG (Vector Laboratories) at 1:200 dilution for 30 min, rinsed again, and mounted with Fluorescence Mounting Medium (DAKO, Glostrup, Denmark). Immunofluorescence images were captured by a 3-CCD color video camera (JVC, Victor Company of Japan LTD, Yokohama, Japan) attached to an Olympus epifluorescence microscope model AX70TF (Olympus Optical Co. LTD, Tokyo, Japan). Image capturing was performed with AcQuis software. All computer-generated pictures were organized by Adobe Photoshop 5.0 software on a PowerPC computer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In cultured HGECs, hBD-2 mRNA expression was induced by high extracellular calcium (0.60 and 1.20 mM) (Fig. 1AGo). The induction was approximately by three- and four-fold in 0.60 and 1.20 mM calcium, respectively, compared with the hBD-2 mRNA expression in HGECs incubated with 0.03 mM calcium (Fig. 1BGo). In contrast, hBD-1 and hBD-3 mRNA expression was not affected by high calcium (Fig. 1AGo). GAPDH served as a control and showed equivalent amounts of RNA between samples (Fig. 1AGo). Similarly, thapsigargin, an endoplasmic reticulum Ca2+ ATPase inhibitor, known to release calcium from internal stores, up-regulated hBD-2 mRNA expression in a dose-dependent manner (Fig. 1CGo). HBD-2 mRNA expression was induced by approximately three-, five-, and six-fold upon incubation with 10, 100, and 1000 nM thapsigargin, respectively, in comparison with hBD-2 mRNA expression in control HGECs (Fig. 1DGo). However, hBD-1 and hBD-3 mRNA expression was not affected by thapsigargin (Fig. 1CGo). IL-8 mRNA was induced by both extracellular calcium and thapsigargin in a dose-dependent fashion (Figs. 1A and 1CGo, respectively). This was consistent with the findings in human colonic epithelial cells (Yu et al., 2001).



View larger version (39K):
[in this window]
[in a new window]
  		Figure 1. The effect of increased extracellular and intracellular calcium on ß-defensin mRNA expression. (A) RT-PCR analysis. HGECs were incubated in various calcium concentrations (0.03, 0.06, 0.15, 0.60, or 1.20 mM) overnight. Total RNA was isolated and RT-PCR was conducted as described in MATERIALS & METHODS. The sizes of the amplified products for hBD-1, -2, -3, IL-8, and GAPDH are indicated and were as predicted. Note that hBD-2 and IL-8 mRNA were induced by increased extracellular calcium concentration in a dose-dependent manner, while hBD-1 and hBD-3 mRNA expression was not affected. A -RT sample was a negative control where enzyme was omitted. The data shown are representative of 5 separate experiments. (B) Densitometric analysis of hBD-2 expression as a function of calcium concentration. The relative ratios of hBD-2 to GAPDH were determined as described in MATERIALS & METHODS. The y axis represents the relative hBD-2 mRNA expression for the different calcium concentrations as shown in panel A. The results are represented as means plus standard deviations of 5 separate experiments. (C) RT-PCR analysis. HGECs were stimulated with various thapsigargin concentrations (0, 1, 10, 100, or 1000 nM) overnight. Note that hBD-2 and IL-8 mRNA were induced by thapsigargin in a dose-dependent fashion, whereas hBD-1 and hBD-3 mRNA expression was not affected. The data shown are representative of 4 separate experiments. (D) Densitometric analysis of hBD-2 expression as a function of thapsigargin concentration as shown in panel C. The results are represented as means plus standard deviations of 4 separate experiments.

 
The time-course study demonstrated delayed (12 hrs) and rapid (1–3 hrs) hBD-2 mRNA induction in response to stimulation with 1.20 mM calcium and 1000 nM thapsigargin (Figs. 2A and 2BGo, respectively). Densitometric analyses revealed an increase in hBD-2 expression by two-fold after HGECs were stimulated with thapsigargin within the first 3 hrs, while more delayed hBD-2 induction, i.e., after 12 hrs of stimulation, was seen in HGECs stimulated with calcium (Fig. 2CGo). Whereas the rapid induction by thapsigargin is similar to that by the cell wall extract of F. nucleatum (2–4 hrs), the delayed induction by extracellular calcium is similar to that by the phorbol ester (10 hrs) (Krisanaprakornkit et al., 2000). This kinetics profile supports our previous findings that suggested involvement of multiple signaling pathways, including 3 MAP kinase pathways, in hBD-2 regulation (Krisanaprakornkit et al., 2002).



View larger version (37K):
[in this window]
[in a new window]
  		Figure 2. Kinetics of hBD-2 mRNA up-regulation by calcium and thapsigargin. HGECs were stimulated with 1.20 mM extracellular calcium (A) or 1000 nM thapsigargin (B) for the indicated times. RT-PCR analysis was performed as described in MATERIALS & METHODS. (C) Densitometric analysis of RT-PCR in panels A and B. The relative ratios of hBD-2 to GAPDH were determined as described in MATERIALS & METHODS. The y axis represents the ratios; the x axis represents various incubation periods (hrs) with either calcium (empty bars) or with thapsigargin (filled bars). Note the rapid and delayed hBD-2 mRNA induction by thapsigargin and high calcium, respectively. The results shown are representative of 3 independent experiments.

 
A recent study has shown that lipopolysaccharide (LPS)-induced hBD-2 expression in a human airway cell line is dependent on intracellular calcium, since an intracellular calcium chelator diminished the effect of LPS on hBD-2 gene expression (Tomita et al., 2002). To determine whether the induction of hBD-2 in oral epithelial cells is dependent on an elevated intracellular calcium, we pre-treated HGECs with various doses of BAPTA-AM for 45 min prior to stimulation with either F. nucleatum cell wall extract for 6 and 12 hrs, calcium, or thapsigargin for 12 hrs. As shown in Fig. 3Go, the addition of cell-permeable BAPTA-AM, which chelates intracellular calcium, inhibited hBD-2 mRNA induction in response to 3 hBD-2 activators in a dose-dependent manner. These results clearly demonstrate that the induction of hBD-2 mRNA in response to bacterial components, high calcium concentration, and thapsigargin is in part dependent on elevation of intracellular calcium by the release of calcium from intracellular stores.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 3. HBD-2 mRNA induction by F. nucleatum is dependent on an increase in intracellular calcium. HGECs were pre-incubated with 0, 10, or 30 µM BAPTA-AM for 45 min, and then stimulated with either 10 µg of F. nucleatum cell wall extract/mL for 6 and 12 hrs, 1000 nM thapsigargin, or 1.20 mM calcium for 12 hrs, or were left as unstimulated controls. RT-PCR analysis was performed as described in MATERIALS & METHODS. Note that hBD-2 induction by all 3 inducers was completely inhibited by 30 µM BAPTA-AM.

 
Immunofluorescence showed hBD-2 peptide in the cytoplasm of cultured HGECs stimulated with 10 µg of F. nucleatum/mL, 1.20 mM calcium, and 1000 nM thapsigargin overnight (Figs. 4B, 4D, and 4EGo, respectively). The staining revealed a punctate distribution of hBD-2 peptide in the cytoplasm (arrows). HBD-2 peptide was not detected in every cell (arrowheads), possibly because of variations in staining of individual cells or because hBD-2 peptide is synthesized in a subpopulation of HGECs. Control unstimulated HGECs showed no immunoreactivity against hBD-2 peptide (Fig. 4AGo). F. nucleatum-stimulated HGECs incubated with pre-immune rabbit serum as a negative control showed no reactivity (Fig. 4CGo).



View larger version (94K):
[in this window]
[in a new window]
  		Figure 4. Localization of hBD-2 peptide in stimulated HGECs. HGECs grown on coverslips were unstimulated (A) or stimulated overnight with 10 µg of F. nucleatum cell wall extract/mL (B,C), 1.20 mM extracellular calcium (D), or 1000 nM thapsigargin (E). HGECs were fixed and reacted with polyclonal antibody against hBD-2 (A,B,D,E) or pre-immune rabbit serum (C), and fluorescein isothiocyanate-conjugated secondary antibody as previously described (Krisanaprakornkit et al., 2000). Bars represent 40 µm. The data shown are representative of 2 separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This work explores signaling pathways that regulate ß-defensin expression. We have previously shown that expression of both hBD-1 and hBD-2 is associated with differentiation in oral epithelia (Krisanaprakornkit et al., 2000; Dale et al., 2001), and that hBD-2 up-regulation by the F. nucleatum cell wall requires activation of MAP kinase pathways in oral epithelial cells (Krisanaprakornkit et al., 2002). In the present study, we provide evidence that hBD-2 expression in oral epithelial cells is also regulated by calcium, utilizing either high extracellular calcium or intracellular calcium stores released by the endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin. In particular, we demonstrated that calcium and thapsigargin stimulated hBD-2 mRNA expression in a dose-dependent fashion and that they induced hBD-2 peptide expression. These in vitro data are consistent with the distribution of hBD-2 mRNA and peptide in the suprabasal and granular layers of the epithelium (Dale et al., 2001), where several markers for terminal differentiation are expressed, and a functional barrier, which is regulated by increased calcium concentration, is formed (Lee et al., 1992). High calcium levels of 0.60 and 1.20 mM, used to induce hBD-2 expression in this study, permit more rapid stratification and formation of cornified cell envelopes and up-regulate several genes involved in terminal differentiation of epithelium and epidermis, e.g., profilaggrin, transglutaminase, and involucrin (Eckert et al., 1997). Moreover, we provide evidence that an increase in intracellular calcium is essential for bacterially induced hBD-2 mRNA expression, since BAPTA-AM also blocked hBD-2 up-regulation by the F. nucleatum cell wall. Calcium also stimulated expression of the chemokine IL-8 in this system. Therefore, calcium-dependent signal transduction in oral epithelial cells is involved not only in the regulation of normal differentiation, but also in the development of mucosal immunity by stimulating hBD-2 and IL-8.

Our findings are consistent with hBD-2 mRNA regulation by calcium in human primary skin keratinocytes (Liu et al., 2002) and with IL-8 mRNA regulation in human colonic epithelial cells (Yu et al., 2001), but extend these findings to show that hBD-2 up-regulation is not only stimulated by extracellular calcium but is also dependent on elevated intracellular calcium. However, our results differ from previous findings (Frye et al., 2001), which showed that increased extracellular calcium in a keratinocyte cell line induced up-regulated expression of hBD-1 mRNA, but not hBD-2 mRNA. The discrepancy may be due to the keratinocyte cell line used in their study, which differs in many respects from the primary keratinocytes used in this study. In addition, their studies were conducted in confluent cultures (as much as 7–14 days post-confluent), while ours were in cultures that were only 80% confluent.

In contrast to hBD-2 induction, hBD-1 and hBD-3 mRNA expression was not significantly affected by increased calcium concentrations, although we have not examined the peptide levels for these ß-defensins. We have previously shown the association between hBD-1 peptide and differentiation in vivo, although the mRNA is detectable in several more epithelial layers than is the peptide (Dale et al., 2001). However, until there is an antibody to the hBD-3 peptide, we cannot examine its localization in the tissue. Furthermore, in vitro data may not completely represent the in vivo situation, because the mechanisms of epithelial differentiation in vivo are far more complex than just the one differentiating factor tested here. Studying other molecular factors that promote epithelial differentiation, such as vitamin D (Bikle et al., 2001), will add to our understanding of mechanisms of ß-defensin regulation.

The time-course study shows rapid hBD-2 mRNA induction by thapsigargin that is comparable with that by F. nucleatum cell wall extract (Krisanaprakornkit et al., 2000). This may be because thapsigargin causes an immediate transient rise of intracellular calcium (Jones and Sharpe, 1994). A sustained hBD-2 induction by prolonged incubation with thapsigargin may result from a subsequent calcium influx from the extracellular medium (Tombal et al., 2002) that results in an increase in intracellular free calcium and eventually leads to differentiation (Li et al., 1995). Likewise, a rapid hBD-2 induction by F. nucleatum may result from an immediate transient rise of intracellular calcium, while a sustained hBD-2 induction by prolonged stimulation with F. nucleatum may result from a calcium influx. The importance of elevated intracellular calcium for hBD-2 up-regulation by F. nucleatum is confirmed by the result in Fig. 3Go, in which 30 µM BAPTA-AM, a cell-permeable calcium chelator, completely inhibits either rapid (6 hrs) or delayed (12 hrs) hBD-2 induction. In contrast to the rapid hBD-2 induction, the delayed hBD-2 induction by increased extracellular calcium (12 hrs), comparable with that by phorbol ester (Krisanaprakornkit et al., 2000), may be due to the relatively slow rise in intracellular calcium and subsequent differentiation, since these kinetics are similar to phorbol-ester-induced differentiation in keratinocytes (Yuspa et al., 1983).

In conclusion, we have demonstrated that high extracellular calcium concentrations and thapsigargin can increase hBD-2 gene expression in oral epithelial cells, which is dependent on the elevation of intracellular calcium. Likewise, an oral commensal bacterium like F. nucleatum that interacts at mucosal surfaces can activate hBD-2 expression through a calcium-dependent pathway. Unraveling the multiple mechanisms involved in the regulation of ß-defensins, especially hBD-2, in oral epithelial cells may lead to a better understanding of normal host defense, disease pathogenesis, and the relationships of innate host defenses with epithelial differentiation.


   ACKNOWLEDGMENTS
 
This work was supported by the Thailand Research Fund (grant no. 4580028 to S.K.). The authors thank Ms. Janet R. Kimball for her assistance on manuscript preparation, and Mr. Tongkam Taya and staff at the Faculty of Medicine, Chiang Mai University, for their technical assistance. We are grateful to all staff at the Department of Oral Surgery, Faculty of Dentistry, Chiang Mai University, Thailand. A preliminary report was presented at the 11th Biennial Meeting of the International Association of Oral Pathologists, Singapore, August 5-8, 2002.

Received December 18, 2002; Last revision June 3, 2003; Accepted July 25, 2003


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ali RS, Falconer A, Ikram M, Bissett CE, Cerio R, Quinn AG (2001). Expression of the peptide antibiotics human beta defensin-1 and human beta defensin-2 in normal human skin. J Invest Dermatol 117:106–111.[ISI][Medline]

Bikle DD, Ng D, Tu CL, Oda Y, Xie Z (2001). Calcium- and vitamin D-regulated keratinocyte differentiation. Mol Cell Endocrinol 177:161–171.[ISI][Medline]

Dale BA (2002). Periodontal epithelium: a newly recognized role in health and disease. Periodontol 2000 30:70–78.

Dale BA, Kimball JR, Krisanaprakornkit S, Roberts F, Robinovitch M, O’Neal R, et al. (2001). Localized antimicrobial peptide expression in human gingiva. J Periodontal Res 36:285–294.[ISI][Medline]

Dunsche A, Acil Y, Dommisch H, Siebert R, Schröder JM, Jepsen S (2002). The novel human beta-defensin-3 is widely expressed in oral tissues. Eur J Oral Sci 110:121–124.[ISI][Medline]

Eckert RL, Crish JF, Banks EB, Welter JF (1997). The epidermis: genes on-genes off. J Invest Dermatol 109:501–509.[ISI][Medline]

Frye M, Bargon J, Gropp R (2001). Expression of human beta-defensin-1 promotes differentiation of keratinocytes. J Mol Med 79:275–282.[ISI][Medline]

Fulton C, Anderson GM, Zasloff MA, Bull R, Quinn AG (1997). Expression of natural peptide antibiotics in human skin. Lancet 350:1750–1751.[ISI][Medline]

Harder J, Bartels J, Christophers E, Schröder JM (1997). A peptide antibiotic from human skin (letter). Nature 387:861.[Medline]

Harder J, Bartels J, Christophers E, Schröder JM (2001). Isolation and characterization of human beta-defensin-3, a novel human inducible peptide antibiotic. J Biol Chem 276:5707–5713.[Abstract/Free Full Text]

Jones KT, Sharpe GR (1994). Thapsigargin raises intracellular free calcium levels in human keratinocytes and inhibits the coordinated expression of differentiation markers. Exp Cell Res 210:71–76.[ISI][Medline]

Krisanaprakornkit S, Weinberg A, Perez CN, Dale BA (1998). Expression of the peptide antibiotic human beta defensin 1 in cultured gingival epithelial cells and gingival tissue. Infect Immun 66:4222–4228.[Abstract/Free Full Text]

Krisanaprakornkit S, Kimball JR, Weinberg A, Darveau RP, Bainbridge BW, Dale BA (2000). Inducible expression of human beta-defensin-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 68:2907–2915.[Abstract/Free Full Text]

Krisanaprakornkit S, Kimball JR, Dale BA (2002). Regulation of human beta-defensin-2 in gingival epithelial cells: the involvement of mitogen-activated protein kinase pathways, but not the NF-kappaB transcription factor family. J Immunol 168:316–324.[Abstract/Free Full Text]

Lee SH, Elias PM, Proksch E, Menon GK, Mao-Quiang M, Feingold KR (1992). Calcium and potassium are important regulators of barrier homeostasis in murine epidermis. J Clin Invest 89:530–538.

Li L, Tucker RW, Hennings H, Yuspa SH (1995). Inhibitors of the intracellular Ca2+-ATPase in cultured mouse keratinocytes reveal components of terminal differentiation that are regulated by distinct intracellular Ca2+ compartments. Cell Growth Differ 6:1171–1184.[Abstract]

Liu AY, Destoumieux D, Wong AV, Park CH, Valore EV, Liu L, et al. (2002). Human beta-defensin-2 production in keratinocytes is regulated by interleukin-1, bacteria, and the state of differentiation. J Invest Dermatol 118:275–281.[ISI][Medline]

Mathews M, Jia HP, Guthmiller JM, Losh G, Graham S, Johnson GK, et al. (1999). Production of beta-defensin antimicrobial peptides by the oral mucosa and salivary glands. Infect Immun 67:2740–2745.[Abstract/Free Full Text]

Menon GK, Elias PM (1991). Ultrastructural localization of calcium in psoriatic and normal human epidermis. Arch Dermatol 127:57–63.[Abstract]

Missero C, Di Cunto F, Kiyokawa H, Koff A, Dotto GP (1996). The absence of p21Cip 1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor 5 progression. Genes Dev 10:3065–3075.[Abstract/Free Full Text]

Niyonsaba F, Iwabuchi K, Matsuda H, Ogawa H, Nagaoka I (2002). Epithelial cell-derived human beta-defensin-2 acts as a chemotaxin for mast cells through a pertussis toxin-sensitive and phospholipase C-dependent pathway. Int Immunol 14:421–426.[Abstract/Free Full Text]

Polakowska RR, Goldsmith LA (1991). The cell envelope and transglutaminase. In: Biochemistry, physiology and molecular biology of the skin. Goldsmith LA, editor. Oxford: Oxford University Press, pp. 168-201.

Tombal B, Denmeade SR, Gillis JM, Isaacs JT (2002). A supramicromolar elevation of intracellular free calcium [Ca2+]i is consistently required to induce the execution phase of apoptosis. Cell Death Differ 9:561–573.[ISI][Medline]

Tomita T, Nagase T, Ohga E, Yamaguchi Y, Yoshizumi M, Ouchi Y (2002). Molecular mechanisms underlying human beta-defensin-2 gene expression in a human airway cell line (LC2/ad). Respirology 7:305–310.[ISI][Medline]

Valore EV, Park CH, Quayle A, Wiles KR, McCray PB Jr, Ganz T (1998). Human beta-defensin-1: an antimicrobial peptide of urogenital tissues. J Clin Invest 101:1633–1642.[ISI][Medline]

Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, et al. (1999). Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286:525–528.[Abstract/Free Full Text]

Yu Y, De Waele C, Chadee K (2001). Calcium-dependent interleukin-8 gene expression in T84 human colonic epithelial cells. Inflamm Res 50:220–226.[ISI][Medline]

Yuspa SH, Ben T, Hennings H (1983). The induction of epidermal transglutaminase and terminal differentiation by tumor promoters in cultured epidermal cells. Carcinogenesis 4:1413–1418.[Abstract/Free Full Text]

Yuspa SH, Kilkenny AE, Steinert PM, Roop DR (1989). Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J Cell Biol 109:1207–1217.[Abstract/Free Full Text]

Zhao C, Wang I, Lehrer RI (1996). Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells. FEBS Lett 396:319–322.[ISI][Medline]




This article has been cited by other articles:


Home page
 Int ImmunolHome page
S. Krisanaprakornkit, P. Chotjumlong, P. Kongtawelert, and V. Reutrakul
Involvement of phospholipase D in regulating expression of anti-microbial peptide human -defensin-2
Int. Immunol., January 1, 2008; 20(1): 21 - 29.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
H. Dommisch, W. O. Chung, M. G. Rohani, D. Williams, M. Rangarajan, M. A. Curtis, and B. A. Dale
Protease-Activated Receptor 2 Mediates Human Beta-Defensin 2 and CC Chemokine Ligand 20 mRNA Expression in Response to Proteases Secreted by Porphyromonas gingivalis
Infect. Immun., September 1, 2007; 75(9): 4326 - 4333.
[Abstract] [Full Text] [PDF]

Home page
 J. Dent. Res.Home page
E. Andrian, D. Grenier, and M. Rouabhia
Porphyromonas gingivalis-Epithelial Cell Interactions in Periodontitis.
J. Dent. Res., May 1, 2006; 85(5): 392 - 403.
[Abstract] [Full Text] [PDF]

Home page
 J. Dent. Res.Home page
A. Dongari-Bagtzoglou and P.L. Fidel Jr.
The Host Cytokine Responses and Protective Immunity in Oropharyngeal Candidiasis
J. Dent. Res., November 1, 2005; 84(11): 966 - 977.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
B. E. Menzies and A. Kenoyer
Staphylococcus aureus Infection of Epidermal Keratinocytes Promotes Expression of Innate Antimicrobial Peptides
Infect. Immun., August 1, 2005; 73(8): 5241 - 5244.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
W. O. Chung, S. R. Hansen, D. Rao, and B. A. Dale
Protease-Activated Receptor Signaling Increases Epithelial Antimicrobial Peptide Expression
J. Immunol., October 15, 2004; 173(8): 5165 - 5170.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
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 ISI Web of Science (12)
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.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
IADR Journals Advances in Dental Research ®
Journal of Dental Research ® Critical Reviews (1990-2004)