Sphingosine Kinase 1 regulated HBD-2 induction in oral keratinocytes

Objective: To assess the role of a new kinase, Sphingosine kinase 1 (SPHK1) in the regulation of human beta defensin 2 (HBD-2) in oral keratinocytes.

Methods: Primary human gingival epithelial cells (HGECs) were challenged with heat inactivated Porphyromonas gingivalis and a variety of Toll-Like Receptor (TLR) and G Protein Coupled Receptors (GPCR) agonists for 24 hours with or without SPHK1 and Phospho-inisitol-3 kinase (PI3K) inhibitors. Culture supernatants were analyzed for HBD-2 by ELISA and Westerns were performed for SPHK1, phospho-ERK 1/2 and phospho-p38 MAPK. The expression plasmid containing SPHK1 gene was transiently transfected to monitor HBD-2 induction and siRNA techniques to knockdown SPHK1.

Results: Stimulation with TLR agonists such as heat inactivated P. gingivalis (TLR2 and 4), Pg-LPS (TLR2), LPS (TLR4), Pam3CSK4 (TLR1 and 2), FSL-1 (TLR2 and 6), Poly I:C (TLR3), Imiquimod (TLR7), ssRNA40 (TLR8), ODN (TLR9), TNF-α and IL-1α/β induced HBD-2 in keratinocytes by activating SPHK1. HBD2 was also induced by stimulating with formyl-met-leu-phe (FMLP receptor), PAR1 and PAR2 peptides. However Poly I:C (TLR 3 agonist) induced higher levels of HBD2 compared to all other agonists. Agonist-activated-HBD-2-secretion is dose dependently inhibited by SPHK1 inhibitor and verified by siRNA knockdown of SPHK1 prior to agonist challenge. Over-expression of SPHK1 in keratinocytes induced higher levels of HBD-2 compared to controls. Further, SPHK1 activated PI3K, p38 MAPK and ERK 1/2 to trigger HBD-2 induction and elucidates a potential link between TLR and GPCR signaling.

Conclusion: These results suggest the involvement of SPHK1 in HBD-2 regulation of oral keratinocytes and provide insights into a new signaling pathway which activates PI3K and in turn activates p38 MAPK and ERK 1/2 to induce HBD-2. Supported by grant DE017384 from NIDCR