CXCL12-stimulated PC3-CXCR4.5 cells (no secretion in non- treated cells versus 4.9 pg/ml of CCL20 in CXCL12-treated cells). After 48 hours of incubation, the levels of intra-cellular CCL20 were significantly elevated and higher CCL20 production was observed in CXCL12-stimulated cells, in addition, the secretion of CCL20 was increased. Both synthesized and secreted CCL20 levels increased in response to CXCL12 stimulation, and the cumulative pattern of synthesis and release was observed over time (Fig. 1F). In order to find whether PC3 and PC3-CXCR4.5 cells secrete CCL20 in a CXCR4/CXCL12 dependent manner, we treated the cells with antibodies to CXCL12 and PTX. In PC3 cells, treatment with neutralizing antibodies against CXCL12 or with PTX (alone or in combination with CXCL12) effectively inhibited the secretion of CCL20. Surprisingly, in PC3-CXCR4.5 cells, anti-CXCL12 antibodies and PTX only partially inhibited the secretion of CCL20 whereas PTX failed to inhibit CXCL12- dependent increase of CCL20 (Fig. 1G). CCL20 production from PC3 cells is dependent on CXCR4/ CXCL12 interaction; however, it was not clear whether the expression and secretion of CCL20 from PC3-CXCR4.5 cells was dependent only on CXCR4. In order to determine the role of CXCR4 in the regulation of CCL20 production, we silenced CXCR4 by RNA interference in PC3-CXCR4.5 - prostate cancer cells with high CXCR4 expression. PC3-CXCR4.5 cells were transfected with Dicer-substrate anti-CXCR4 mRNA specific siRNA. The efficacy of CXCR4 silencing was assessed 48 hours post-transfection. We achieved over 90% reduction in CXCR4 cell surface expression (Fig. 2A) and 75% reduction in mRNA levels (Fig. 2A) using siRNA to CXCR4 RNA. Next, we sought to determine whether the reduction in CXCR4 might inhibit CCL20 production in PC3-CXCR4.5 cells. Forty eight hours following transfection with siRNA, PC3-CXCR4.5 cells were re-seeded and stimulated with CXCL12 for an additional 48 hours. CCL20 secretion levels in culture medium of the cells transfected with either nonspecific control siRNA or the CXCR4 siRNA were measured using ELISA assay. Silencing of CXCR4 significantly reduced both constitutive and CXCL12-stimulated levels of CCL20 production. In non-stimulated cells, the secretion of CCL20 was decreased from 350656 pg/ml in control cell to 4262.8 pg/ml in cells with CXCR4 siRNA (p,0.0054). In CXCL12-stimulated cells, the secretion of CCL20 was decreased from 6006113 pg/ml in control cells to 137.5653 pg/ml in cells with CXCR4 siRNA (p,0.0035, Fig. 2A). In addition, we tested the effect of CXCR4 silencing on CCL20 mRNA level using real- time RT-PCR. Silencing of CXCR4 resulted in a 3-fold decrease in CCL20 mRNA level in PC3-CXCR4.5 cells (Fig. 2B). CCL20 expression and secretion in the parental PC3 and PC3- CXCR4.5 cells that overexpress CXCR4 are regulated by CXCR4 (Fig. 1G and Fig. 2A). PC3 cells express low levels of CXCR4 and secrete low levels of CCL20, whereas PC3- CXCR4.5 cells express high levels of CXCR4 and ten-fold higher levels of CCL20. In both cell lines, treatment with PTX or anti CXCL12 blocked CCL20 secretion; however, the inhibitory effect of PTX or anti CXCL12 was much more evident in the parental cells. Similarly, the effect of PTX on CXCL12-induced secretion of CCL20 was partial, albeit significant only in the parental PC3 cells. The differences between these cells may be the result of a more robust activation of diverse signaling pathways activated by CXCR4 and the use of insensitive Ga chain used by CXCR4. Various signal transduction pathways have been implicated through the association of CXCR4 receptors with the guanine nucleotide binding protein (G-proteins). For example, stimulation of CXCR4 transfectants by CXCL12 results in increased CCL20 and CXCR4 Interaction phosphorylation of focal adhesion components, including the related adhesion focal tyrosine kinase (RAFTK/Pyk2), Crk, and paxillin. CXCL12 induced activation of the p44/42 MAP kinases (Erk 1 and 2), PI3 kinase, and NF kB [18,19,20]. Moreover, a short time following CXCL12 activation, the CXCR4 receptor becomes tyrosine phosphorylated through the activation and association with the receptor of JAK2 and JAK3 kinases [21,22]. Several STAT transcriptional factors, including STAT1, -2, -3, and -5, but not STAT4 or -6, are associated with CXCR4 activation, concurring with the role assigned to the JAK tyrosine kinases in transducing signals from hematopoietic growth factor receptors. Tyrosine phosphorylation of two other members of the chemokine receptor family, CCR2 and CCR5, in response to their respective ligands (MCP-1 and RANTES) and the critical role of the JAK/STAT pathway activation in later chemokine signaling events were also documented. The activation in T cells of different STATs by the chemokines RANTES and MIP 1a has also been reported. Tyrosine phosphorylation is not a unique feature of chemokine receptors, since other GPCRs also activate STAT transcription factors. Interestingly, JAK/STAT pathway activa- tion by chemokines and their receptors is not always blocked by PTX pretreatment, indicating a G-protein-independent pathway. To address the importance of JAK/STAT and MEK activation on the secretion of CCL20, we treated the PC3 and PC3- CXCR4.5 cells with JAK-2 inhibitor AG-490 (Cat. 658401; Calbiochem) at 1 mm/ml. and the MEK inhibitor- PD98059 (20 mM) (Calbiochem, San Diego, CA) PD. In both cell lines, the JAK-2 inhibitor AG-490 totally abrogated CCL20 secretion, whereas the MEK inhibitor- PD98059 slightly increased the levels of CCL20. DMSO used for dissolving PD or AG-490 had an inhibitory effect on CCL20 secretion, and more significantly on the parental PC3 cells (Fig. 1G). These results may suggest a role for the JAK/STAT pathway in the CXCL12-induced secretion of CCL20. The increased levels of CCL20 associated with increased levels of CXCR4 do not correlate with the CXCL12 low levels secreted by PC3 or PC3-CXCR4.5 cells and cannot be blocked by anti CXCL12 when added to PC3-CXCR4.5 cell cultures (Fig. 1G). One possibility to explain such a phenomenon is the presence of a CXCR4 ligand other than CXCL12. Although chemokines typically display a high degree of receptor promiscuity, CXCR4 was (until recently) thought to bind only to CXCL12. However, recently, it was demonstrated that the macrophage migration inhibitory factor (MIF) can compete with the recognized ligand for CXCL12 for binding to CXCR4 [23]. CXCR4 was shown to be homo-oligomerized by several experimental systems. It is possible that CXCR4 oligomerization may lead to autonomous signaling which is mediated through JAK/ STAT signaling. To better understand the role of CCL20 in prostate cancer development, we characterized the expression of CCL20 and its receptor CCR6 in human prostate cell lines PC3, LNCaP, 22RV1 and DU145. We first examined CCR6 receptor surface and mRNA expression levels in these four cell lines. RT-PCR analysis and FACS analysis demonstrated that only PC3 cell line expressed CCR6 receptor at mRNA level and on the cell surface (Fig. 2C). Next, we performed ELISA experiments to determine the secretion levels of CCL20 chemokine. Among the four prostate cancer cell lines studied, only PC3 cells secreted detectable levels of CCL20 into the culture supernatant during the 48 hours incubation (Fig. 2C). However, in addition to PC3 cells, the mRNA expression of CCL20 was demonstrable in DU145 cells and at a very low level also in LANCaP cells (Fig. 2C). Since PC3 cells co-expressed the CCR6 receptor and its ligand CCL20, we focused our in vitro and in vivo experimental work on PC3 cells. PLoS ONE | www.plosone.org 3 April 2009 | Volume 4 | Issue 4 | e5125