Dose dependent inhibition of HBD-2 induction in HGECs upon Sphk-1 inhibition.

<p>Keratinocytes were pretreated with Sphk-1 inhibitor at various concentrations ranging from 0.1 µM to 5.0 µM for 2 h prior to challenging the cells with FSL-1 ligand (1 µg/ml) for 24. Supernatant was subjected to human HBD-2 ELISA. Sphk-1 inhibitor dose dependently inhibited the induction of HBD-2 (<b>A</b>). In another set of experiment, total protein was collected after 60 min of challenge as mentioned above. Immunoblot was performed with ser225 phospho specific Sphk-1 antibody and β-actin as loading control. The level of phosphorylation of Sphk-1 at ser225 was dose dependently down regulated upon Sphk-1 inhibitor treatment (<b>B</b>). Results are mean ± SEM and are representative of three independent experiments.</p

RNAi mediated inhibition of Sphk-1 inhibits agonists induced HBD-2.

<p>The cells were transiently transfected with siRNA against Sphk-1 and incubated with FSL-1 (1 µg/ml), Imiquimod (0.1 µg/ml) and IL-1β (2.5 ng/ml). The supernatant was collected after 24 h challenge and subjected to human HBD-2 ELISA. siRNA against Sphk-1 down modulated agonist induced HBD-2 in HGECs (<b>A</b>). Intracellular S1P was measured using S1P ELISA kit after challenging with FSL-1 (1 µg/ml) for 2 h in cells pre-incubated with Sphk-1 inhibitor. The reaction was terminated after 2 h and 100 µg of total protein was subjected to ELISA. The intracellular S1P production was downregulated by Sphk-1 inhibitor (<b>B</b>). Total RNA was collected and converted to cDNA from the cells challenged with FSL-1 (1 µg/ml) for 24 h. cDNA was subjected to real time PCR with Sphk-1, Sphk-2 and GAPDH endogenous control TaqMan probes. Sphk-1 mRNA expression was highly upregulated compared to Sphk2 mRNA expression showing Sphk-1 as a predominant kinase in HGECs upon ligand challenge (<b>C</b>). Control cells received DMSO unless otherwise stated. Results are mean ± SEM and are representative of three independent experiments. Statistical comparisons are shown by horizontal bars with asterisks above them (* indicates p<0.05 determined by ANOVA and Tukey multiple comparison test).</p

Model of mechanism involved in HBD-2 secretion in gingival epithelial cells.

<p>Triggering TLRs by respective ligand stimulates the cells to induce HBD-2. This induction can be ablated by a pharmacological inhibitor against Sphk-1, and inhibition of GSK3 by SB216763 or siGSK3-β in the absence of Sphk-1 inhibition can augment HBD-2 secretion. Inhibition of PI3K by either wortmannin or LY294002 abrogated HBD-2 in gingival epithelial cells. PI3K activated Akt, the phosphorylation of Akt inhibited GSK3 in turn activating ERK 1/2. ERK 1/2 activates Sphk-1 by phosphorylation at Ser225 and increase NF-kB activity. This show PI3K-Akt-GSK3-ERK1/2-Sphk-1 mediates HBD-2 synthesis in gingival epithelial cells.</p

Overexpression of GSK3-β in HGECs.

<p>Cells were transiently transfected with GSK3-β (S9A), GSK3-β (K85) and pCDNA3.1 as Mock using Fugene 6. The transfected cells were challenged with FSL-1 (1 µg/ml) for 24 h. The supernatant was subjected to human HBD-2 ELISA. The cells transfected with kinase dead GSK-3β (K85) plasmid induced significantly higher amounts of HBD-2 however; the cells transfected with constitutively active GSK3-β (S9A) attenuated HBD-2 induction (<b>A</b>). We overexpressed Sphk-1 by transfecting the cells with Sphk-1 plasmid and incubated in the presence or absence of GSK3-β inhibitor SB 216763. The FSL-1 (1 µg/ml) challenged cells induced significantly higher amounts of HBD-2 compared to the cells without GSK3 inhibition (<b>B</b>). NF-kB p65 activity was measured in the presence or absence of Sphk-1 and GSK3 inhibitor. Sphk-1 inhibition reduced NF-kβ activity however, NF-kβ activity increased upon GSK3 inhibition when challenged with FSL-1 (1 µg/ml) (<b>C</b>). The cells were pretreated with SB216763 for 2 h prior to challenging with FSL-1 for 24 h and supernatant was subjected to ELISA using appropriate kits. IL-1β, TNFα and IL-6 were significantly upregulated upon GSK3 inhibition in the presence of FSL-1 (<b>D</b>). Control cells received DMSO unless otherwise stated. Results are mean ± SEM of triplicates and are representative of three independent experiments. Statistical comparisons are shown by horizontal bars with asterisks above them (* indicates p<0.05 determined by ANOVA and Tukey multiple comparison test).</p

Inhibition of GSK3 augments HBD-2 induction.

<p>Keratinocytes were pretreated with PI3K inhibitor (LY294002–10 µM and wortmannin –0.5 µM) or Akt inhibitor (10 µM) or MEK 1/2, U0126 (25 uM) or GSK3 inhibitor (SB216763-12 µM) 2 h before challenging with FSL-1(1 µg/ml) for 24 h. The supernatant was subjected to human HBD-2 ELISA. PI3K, Akt and MEK inhibitors abrogated HBD-2 induction whereas GSK3 inhibitor augmented HBD-2 induction in HGECs (<b>A</b>). The cells were pretreated with LY294002 (10 µM) for 2 h and challenged with FSL-1 (1 µg/ml) for 0, 30, 60, 90, 120 and 240 min. Total protein was subjected to immunoblot against phospho- ser225 Sphk-1 antibody with β-actin as loading control. PI3K inhibitor downregulated the phosphorylation level of Sphk-1 at Ser225 demonstrating the pivotal role of PI3K in Sphk-1 activation (<b>B</b>). The cells were transient transfected of siRNA against GSK-3β and challenged with FSL-1 (1 µg/ml) for 24 h and supernatants were subjected to human HBD-2 ELISA. siGSK3-β up regulated HBD-2 induction after challenging with FSL-1. This upregulation of HBD-2 was significantly higher to FSL-1 challenge alone (<b>C</b>). The cells were either pretreated with U0126 (25 uM) and/or SB216763 (12 µM) prior to challenging with FSL-1 (1 µg/ml) for 24 h and supernatant was subjected to HBD-2 ELISA. GSK3 inhibitor augmented the HBD-2 induction whereas the cells with GSK3 +MEK 1/2 inhibitor ablated HBD-2 induction (<b>D</b>). Phospho-Glycogen synthase (Ser641) levels were assessed by incubating SB216763 (12 µM) for 2 h prior to challenge with FSL-1 for 0, 30 and 60 min. Total protein was subjected to immunoblot against Phospho-Glycogen synthase (Ser641) antibody with β-actin as loading control. The phosphorylation of Glycogen synthase at Ser641 was down regulated in the presence of SB216763 (<b>E</b>). Time course experiment was performed either in the presence or absence of SB 216763 inhibitor (GSK3) (12 µM). The cells were challenged with FSL-1 (1 µg/ml) and the total protein was collected at 15, 30 and 60 min and subjected to immunoblot against phospho- ser225 Sphk-1 antibody and p44/42 MAPK (Erk1/2) antibody with β-actin as loading control. Inhibition of GSK3 by SB 216763 increased the Sphk-1 phospho-ser225 at 60 min, ERK 1/2 phosphorylation increased at 30 min of agonist challenge (<b>F</b>). The phosphorylation of p44/42 MAPK (Erk1/2) was unaltered upon Sphk-1 inhibitor after 60 min demonstrating Sphk-1 downstream of Erk 1/2 (<b>G</b>). Control cells received DMSO unless otherwise stated. Results are mean ± SEM and are representative of three independent experiments. Statistical comparisons are shown by horizontal bars with asterisks above them (* indicates p<0.05 determined by ANOVA and Tukey multiple comparison test).</p

Sphk-1 inhibition down modulates agonists induced HBD-2.

<p>Oral keratinocytes were incubated with or without Sphk-1 inhibitor (2 µM) for 2 h prior to challenging the cells with various TLR agonists namely, heat inactivated <i>P. gingivalis</i> (MOI:100), FSL-1 (1 µg/ml), Pam3CSK4 (0.5 µg/ml), <i>P.gingivalis</i> LPS (1 µg/ml), <i>E. Coli</i> LPS (1 µg/ml), ssRNA (0.1 µg/ml), Poly I:C (5 µg/ml) ODN (0.5 µg/ml), Imiquimod (0.1 µg/ml), Flagellin (0.25 µg/mL) (<b>A</b>); the IL-1α (2.5 ng/ml), IL-1β (2.5 ng/ml) and TNF-α (2.5 ng/ml) (<b>B</b>) and GPCR agonist S1P (100 nM) (<b>C</b>) for 24 h. The supernatant was collected after 24 h and HBD-2 ELISA was performed using Human BD-2 ELISA kit. The Sphk-1 inhibitor ablated HBD-2 induction with the agonists tested. The time course experiment was performed by pretreating the cells with Sphk-1 (2 µg/ml) for 2 h before challenging with FSL-1 (1 µg/ml) for 0, 30, 60, 90, 120 and 240 min. The total protein was collected and subjected to immunoblot with ser225 phospho specific Sphk-1 antibody and β-actin as loading control. We noted increase in the phosphorylation level of Sphk-1 at ser225 as early as 60 min and the level of phosphorylation was down regulated in the presence of Sphk-1 inhibitor (<b>D</b>). Control cells received DMSO unless otherwise stated. Results are mean ± SEM and are representative of three independent experiments. Statistical comparisons are shown by horizontal bars with asterisks above them (* indicates p<0.05 determined by ANOVA and Tukey multiple comparison test).</p

<i>P. gingivalis</i> up-regulation of keratinocyte differentiation markers is FOXO1 or FOXO3 dependent.

<p>Primary human gingival epithelial cell cultures were incubated with or without <i>P. gingivalis</i> at MOI = 1∶50 for 20 hrs. In some cases cells were pre-incubated with FOXO1 siRNA (SiFOXO1), FOXO3 siRNA (SiFOXO3) or scrambled siRNA (SiScr) FOXO1 prior to stimulation with bacteria. Real-time PCR was used to measure mRNA levels of keratin-1, keratin-10, involucrin and keratin-14. * Significantly different from control cells without bacterial stimulation (P<0.05). ** Significantly different between scrambled siRNA and FOXO1 or FOXO3 siRNA (P<0.05). + Significantly different between FOXO1 siRNA and FOXO3 siRNA (P<0.05).</p

<i>P. gingivalis</i> but not <i>F. nucleatum</i> or <i>S. gordonii</i> induces expression of differentiation markers.

<p>Primary human gingival epithelial cell cultures were incubated with or without exposure to an air-liquid-interface to induce differentiation and then challenged with <i>P. gingivalis</i> (Pg), S. <i>gordonii</i> (Sg), or <i>F. nucleatum</i> (Fn) at 2×10⁸/cm² for 24 hrs. mRNA levels of keratin-1 or keratin-10 were measured by real-time PCR. +Significant difference between undifferentiated and differentiated cells (P<0.05). * Significantly different from matched control (P<0.05).</p

<i>P. gingivalis</i> induces gingival epithelial cell apoptosis through FOXO1 or FOXO3.

<p>Primary human gingival epithelial cells were incubated with or without P. gingivalis at MOI = 1∶10 or 1∶50 for overnight. In some cases cells were pre-incubated with FOXO1 siRNA (SiFOXO1), FOXO3 siRNA (SiFOXO3) or scrambled siRNA (SiScr) prior to incubation with bacteria. Apoptotic cells were assessed by the TUNEL assay. * Significantly different from control cells with bacterial stimulation (P<0.05). ** Significantly different between scrambled siRNA and FOXO1 or FOXO3 siRNA (P<0.05).</p

<i>P. gingivalis</i> up-regulates FOXO1 and FOXO3 mRNA levels.

<p>Primary human gingival epithelial cell cultures were incubated with or without <i>P. gingivalis</i> at MOI = 1∶50 for 20 hrs. In some cases cells were pre-incubated with FOXO1 siRNA (SiFOXO1), FOXO3 siRNA (SiFOXO3) or scrambled siRNA (SiScr) FOXO1 prior to stimulation with bacteria. FOXO1 and FOXO3 mRNA levels were measured by real-time PCR. * Significantly different from control cells without bacterial stimulation (P<0.05). ** Significantly different between scrambled siRNA and FOXO1 or FOXO3 siRNA (P<0.05). + Significantly different between FOXO1 siRNA and FOXO3 siRNA (P<0.05).</p