Table 1.
Primer sequences and RT-PCR conditions.
Fig 1.
(A) A semiquantitative RT-PCR analysis was performed on the immortalized human prostate epithelial cell line (RWPE-1) and the human prostate cancer cell lines (PC-3 and DU145). (B) Semiquantitative RT-PCR analysis of differential DEFB131 mRNA expression after RWPE-1 cell exposure to various TLR agonists. (C) Semiquantitative RT-PCR analysis of DEFB131 mRNA expression in RWPE-1 cells after treatment with LTA (1 μg/mL). ACTB was used as an internal control.
Fig 2.
TLR2 is required for LTA-induced DEFB131 upregulation in RWPE-1 cells.
(A) Semiquantitative RT-PCR analysis for differential expression of TLR2, TLR6, and CD14 in various human prostate cell lines. THP-1, a human monocyte cell line, was used as the positive control. ACTB was used as an internal control. (B) TLR2 gene expression in TLR2 shRNA-transfected RWPE-1 cells was determined using semiquantitative RT-PCR and western blot analyses. (C) LTA stimulates DEFB131 through TLR2 in RWPE-1 cells. The DEFB131 concentration was measured by ELISA. Concentrations are represented as pg of protein per mL of the supernatant and are representative of five distinct experiments. Different letters (a and b) indicate significant differences at P < 0.005 between groups. (D) An immunocytochemistry analysis was carried out to confirm the data obtained from the ELISA analysis. RWPE-1 cells were stained with anti-DEFB131 antibody (Green) and counterstained with propidium iodide (Red) for DNA.
Fig 3.
Induction of DEFB131 gene expression by LTA is p38MAPK- and NF-κB-dependent.
(A) Phosphorylation of p38MAPK and IκBα determined by western blot after LTA stimulation. LTA stimulated an increase in p38MAPK and IκBα phosphorylation in RWPE-1 cells. In contrast, expression of IκBα was significantly reduced in a time-dependent manner. (B) Inhibition of p38MAPK and NF-κB blocks DEFB131 production. ELISA analysis showed that SB203580 (p38MAPK inhibitor) and Bay11-7082 (NF-κB inhibitor) inhibited secretion of LTA-induced DEFB131 protein. Asterisks (* and **) represent statistical significance at P < 0.0001 and P < 0.002. (C) Immunocytochemistry of DEFB131 after SB203580 or Bay11-7082 treatment showed a reduction in DEFB131 protein expression.
Fig 4.
NF-κB binds to the DEFB131 gene promoter.
(A) Five potential NF-κB/p65 binding sequences are located in the human DEFB131 promoter region. The differences in the site sequences from the consensus NF-κB binding sequences (CBS) are indicated by asterisks. CBS, GGGRNNYYCC or HGGARNYYCC; R, purine; Y, pyrimidine; H, A, C, or T. (B) Chromatin immunoprecipitation assays with antibody directed against NF-κB were performed on RWPE-1 cells treated with LTA. Different letters indicate significant differences between groups at P < 0.05 or P < 0.005.
Fig 5.
DEFB131 induces cytokine and chemokine expression in RWPE-1 cells.
(A) DEFB131 mRNA upregulation. RWPE-1 cells were transfected with an empty vector or the DEFB131-DDK-Myc vector. Quantitative real-time PCR analysis was used to determine mRNA levels. Expression levels were calculated from CT values and normalized to ACTB. Asterisks represent statistical significance at P < 0.0001. (B) Upregulation of DEFB131 protein. DEFB131 protein levels were analyzed by western blot in cell lysates. (C) Overexpression of secreted DEFB131 protein by western blot in culture supernatants. (D, E) Gene expression profiles for cytokines and chemokines in DEFB131-activated RWPE-1 cells were determined using quantitative real-time PCR analysis. Asterisks (* and **) represent statistical significance at P < 0.01 and P < 0.05.
Fig 6.
DEFB131 enhances cytokine and chemokine secretion in the culture supernatants of RWPE-1 cells.
Supernatants obtained from RWPE-1 cells transfected with an empty vector or DEFB131-DDK-Myc were collected after 24 h. The levels of cytokines and chemokines released into the medium were quantified by multi-analyte ELISA. Error bars represent the standard error of the mean (SEM). Asterisks (* and **) represent statistical significance at P < 0.001 and P < 0.01.
Fig 7.
DEFB131 enhances chemotactic activity in monocytic THP-1 cells.
Chemotactic activity was determined by measuring THP-1 migration in response to DEFB131-conditioned cell culture supernatant. The results are presented as a migration index denoting the fold-increase in cell migration as compared to the migration of cells transfected with the empty vector. MCP-1 (100 ng/mL) was used as a positive control. Results are representative of three independent experiments. Asterisks represent statistical significance at P < 0.01.
Fig 8.
DEFB131 affects innate and adaptive immune responses in human prostate epithelial cells.
LTA binds to the TLR2/TLR6 receptor complex and activates AP1 and NF-κB transcription factors through stimulation of the p38MAPK and NF-κB pathways in RWPE-1 cells. Upon LTA stimulation, the activated transcription of AP1 and NF-κB markedly induces DEFB131 expression and secretion, followed by increased production of cytokines and chemokines. Secreted DEFB131 exhibits antimicrobial activity. DEFB131 induces cytokine and chemokine expression and subsequent immune cell recruitment. DEFB131 may contribute to the activation of prostate epithelial cell innate and adaptive immune responses.