CCL20 and CXCR4 Interaction Figure 1. CXCR4 up-regulates CCL20 production by prostate cancer cell line PC3. (A–C) Wild-type PC3 cells, CXCR4-transduced PC3 cell line and single cell clone with stable overexpression of CXCR4 (PC3-CXCR4.5) were stained for the control (IgG2a-PE, purple) and CXCR4 antibodies (IgG2a-12G5, green) and analyzed using FACS. PC3 and PC3-CXCR4.5 cells were stimulated with CXCL12 at concentrations of 50, 250 and 1,000 ng/ml for 48 hours, harvested and viable cells were counted using PI staining and FACS analysis. PC3 and PC3-CXCR4.5 cells were (56106/mice) were injected subcutaneously into SCID/beige mice. 60 days following the injection, animals were sacrificed, tumor size (cm2) and tumor weight (g) were measured. Data is presented as mean6SE from 5 mice. (D) PC3 and PC3-CXCR4.5 cells were stimulated with CXCL12 at concentrations 5 and 500 ng/ ml for 24 hours, total RNA was extracted, reverse-transcribed and subjected to quantitative PCR for CCL20. PCR analysis was carried out in triplicates. (E) PC3 and PC3-CXCR4.5 cells were stimulated with various concentrations of CXCL12 (5, 50, 25, 500, and 1,000 ng/ml) for 48 hours and CCL20 secretion was assessed by ELISA. The results represent the average of triplicates6SD (** P,0.05). (F) PC3-CXCR4.5 cells were incubated with CXCL12 at concentration of 500 ng/ml. At the indicated time points CCL20 was assessed in extra-cellular (culture medium) and intra-cellular (whole cell lysate) fractions using ELISA method. (G) In order to inhibit CXCR4 signaling, PC3 and PC3-CXCR4.5 cells were cultured with anti-CXCL12 antibodies or pertussis toxin alone or in combination with CXCL12 during 48 hours, and CCL20 secretion was assessed by ELISA. CCL20 secretion in PC3 and PC3- CXCR4.5 cells was also inhibited using JAK-2 inhibitor AG-490 at 1 mm/ml. and the MEK inhibitor- PD98059 (20 mM). The results represent the average of triplicates6SD (** P,0.05). doi:10.1371/journal.pone.0005125.g001 further study the possible role of CXCR4 in the regulation of CCL20 expression, we evaluated the mRNA expression of CCL20 in prostate cancer cells with low and high CXCR4 expression using real-time RT-PCR. We found a 41-fold increase in CCL20 mRNA in cells with high CXCR4 expression compared to the wild-type PC3 cells. Moreover, stimulation of both PC3 and PC3- CXCR4.5 cells with CXCL12, the ligand of CXCR4, increased CCL20 mRNA levels in a dose-dependent manner (Fig. 1D). Next, we assessed whether the constitutive and regulated expression of CCL20 mRNA was paralleled by protein produc- tion, using an ELISA assay to CCL20. Corresponding with PCR results, CXCR4 over-expression in PC3 cells increased the constitutive levels of CCL20 secretion from 8.664.5 pg/ml in PC3 cells to 433630 pg/ml in PC3-CXCR4.5 cells (Fig. 1E). Moreover, the stimulation of both parental PC3 and PC3- CXCR4.5 cells with CXCL12 increased the secretion of CCL20 in a dose-dependent manner (Fig. 1E). To determine the pattern of CCL20 release, we tested the protein levels of CCL20 in a culture medium and in lysed cells following different periods of incubation with CXCL12. PC3-CXCR4.5 cells were incubated with CXCL12 500 ng/ml for 6, 24 or 48 hours; culture medium was collected and the cells were lysed with lysis buffer containing 1% NP-40. Stimulation with CXCL12 increased the intracellular levels of CCL20 following 6 hours of incubation, however there was no release of CCL20 to the culture medium at this time point. Following 24 hours of incubation, intra-cellular levels of CCL20 in non-stimulated and in CXCL12-stimulated cells were increased, as well the secreted CCL20 was detected in the culture medium of PLoS ONE | www.plosone.org 2 April 2009 | Volume 4 | Issue 4 | e5125