Clin. (JNK), and p38 and stimulated with dental commensal and and a cell wall structure extract from the dental commensal (11, 17, 23, 24, 29). Up-regulation of hBD-2 in HOK with the cell wall structure included mitogen-activated protein (MAP) kinase signaling pathways however, not NF-B transcription elements (23). The NF-B transcription aspect pathway is essential in the mobile response to inflammatory stimuli also to the entire response to pathogens in lots of cell types, mediating signaling from Toll-like receptors (TLRs) in response to microbial ligands. Our objective was to characterize additional the signaling pathways involved with hBD-2 induction in response to commensal and pathogenic bacterias through the use of both dental and epidermis keratinocytes. We hypothesized that epithelial cells react differently to commensal and pathogenic bacteria and that different signaling pathways are involved in hBD-2 up-regulation by commensal and pathogenic bacteria. In this study, we distinguished the utilization of these pathways by using specific inhibitors of each pathway and verified by quantitative methods which pathways are essential for hBD-2 induction. We provide evidence that different bacteria utilize different pathways for hBD-2 induction, and a common pattern that was observed suggests that commensals and pathogens may utilize different pathways for inducing hBD-2. MATERIALS AND METHODS Human epithelial cells and bacterial culture conditions. Healthy gingival samples were obtained from patients undergoing third-molar extraction at the Department of Oral Surgery, School of Dentistry, University of Washington. Fresh human neonatal foreskin samples were collected from the Dermatology Clinic at the University of Washington Medical Center. Tissue was cut into small pieces (2 by 2 mm) and treated with 6 mg of Dispase (Becton Dickinson, Franklin Lakes, N.J.)/ml overnight at 4C to separate the epithelium from the underlying fibrous connective tissue. The epithelium readily dissociated and was incubated at 37C in Dibutyl phthalate 5 ml of trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA) for 10 min. Subsequently isolated HOK and HFK were grown to 80% confluence in KBM supplemented with KGM (Cambrex, Walkersville, Md.). (ATCC 33277) cells were cultured under anaerobic conditions (85% N2, 10% H2, 5% CO2) at 37C in Trypticase soy broth (BBL, Sparks, Md.) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter. and were grown in Trypticase soy broth at 37C under static conditions. was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37C under anaerobic conditions. Bacterial numbers were determined by measuring density with a Klett-Summerson photometer. Inhibitors. Table ?Table11 lists the inhibitors used to block specific signaling pathways. The concentrations used for 1-pyrrolidinecarbodithioic acid (PDTC), MG132, and SB203580 were determined based on an earlier study (23). For c-Jun N-terminal kinase (JNK) I (JNKI) Dibutyl phthalate and JNKII, doses of 50 Dibutyl phthalate nM, 100 nM, 1 M, and 10 M were tested for their ability to block hBD-2 induction in HOK by the cell wall; 1 M was determined to be the concentration that efficiently blocked hBD-2 induction without cytotoxicity. TABLE 1. Specific inhibitors used in this study DNA polymerase. The PCR conditions were denaturation at 94C for 30 s, annealing at 57C for 30 s, and elongation at 72C for 2 min for 35 cycles. A housekeeping gene, that for ribosomal protein (RPO), was used as a control to determine the total RNA level. The oligonucleotides for hBD-2 and RPO were previously described TLN2 (24). Conditions for real-time PCR. The resulting cDNA was analyzed by using an iCycler (Bio-Rad, Hercules, Calif.) and a QuantiTech SYBR green PCR kit (Qiagen, Valencia, Calif.) according to the manufacturers’ suggestions. The SYBR green mixture contained 100 mM KCl, 40 mM Tris-HCl (pH 8.4), 0.4 mM each dNTP, 50 U of DNA polymerase/ml, 6 mM MgCl2, SYBR green I, and 20 nM fluorescein. The reaction was set up in a 96-well plate, with Dibutyl phthalate each well containing 25 l of the SYBR green mixture, 5 l of cDNA, and 250 nM each primer. The amplification conditions were initial denaturation at 95C for 15 min followed by 40 cycles of denaturation at 95C for 15 s, annealing at 57C for 15 s, and elongation at 72C for 30 s. Melting curve analysis was performed in order to confirm that the detected signal was that of SYBR green binding to the expected amplification product and not to the possible primer-dimers. The amplified product was run on an agarose gel to confirm that.