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

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;

View larger version (39K): [in this window] [in a new window]   Figure 1. The effect of increased extracellular and intracellular calcium on β-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.

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.

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.

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.

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