PKCf Induced by HGF Increases CXCR4 Expression Figure 4. Overexpressed CXCR4 in HGF-stimulated MDA-MB-436 cells is functional. (A–B). Confluent cells were grown in 0.5% FBS medium for 24 hours and were then wounded with a tip. The cells were washed, and the medium was replaced with or without addition of HGF, PSf peptides or AMD3100. Representative micrographs of the wounds are shown together with the results of the migration quantification. Results are presented as the mean 6 SD of 3 independent experiments. # P,0.01 as compared to PBS. Original magnification, 2006. (C).Dose and time- dependent response of HGF-induced MDA-MB-436 cell migration. (D).Dose-dependent response of HGF-induced MDA-MB-436 cell chemotaxis. (E).PKC and CXCR4 regulate HGF-mediated chemotaxis. MDA-MB-436 cells were incubated for 30 minutes with 10 mM chelerythrine chloride, an inhibitor of all PKC, or for 1 hour with 10 mM of PS-a/b, PS-e, or PSf peptide. (F). Boyden chamber assays were performed using the SDF-1 ligand for CXCR4 as a chemotactic attractive agent in the lower chamber. AMD3100, PSf, PSa/b, PSe and NSC23766 (upper) or PKCf-siRNA (lower) was added to the cell culture for the blocking assay. Data are shown as the mean 6 SD of three experiments. A representative study is shown. (G). qRT-PCR analysis of the MT1-MMP mRNA extracted from MDA-MB-436 cells cultured for 24 hours with the indicated agents. Results are presented as the mean 6 SD of three independent experiments. # P,0.01 as compared to PBS. (H). Western blot analysis of the total protein expression levels of MT1-MMP in MDA- MB-436 cells cultured for 24 hours with the indicated agents. The experiment was repeated three with similar results. A representative study is shown. doi:10.1371/journal.pone.0029124.g004 (Figure 6C). This result was further quantified by qRT-PCR for human HPRT, which demonstrated that HGF increased the number of metastatic breast cancer cells in the lung and liver of MDA-MB-436 xenograft-bearing mice by about 7- and 4-fold, respectively, as compared with PBS injection (Figure 6D). Collectively, these data suggested that HGF enhanced the metastasis of breast cancer xenografts to the lung and liver. HGF injection failed to enhance the peritumoral penetration of MDA-MB-436 xenografts infected with PKCf-shRNA but not GFP-shRNA (Figure 6A, Figure S8). Moreover, the prometastatic effect of HGF on MDA-MB-436 xenografts was tremendously alleviated by infection with PKCf-shRNA but not GFP-shRNA (Table S2). Immunoblot and immunohistochemistry with the anti- phospho-PKCf or anti-phospho-CXCR4 antibody further con- firmed that PKCf-shRNA reduced the expression of phosphory- lated PKCf and HGF-induced phosphorylated CXCR4 in cancer cells of the MDA-MB-436 xenografts (Figures 6E–6G, Figures S6,S7). These findings shed light on the HGF-enhanced invasiveness of cancer cells via PKCf in tumor-bearing mice. Discussion The present study addressed the molecular aspects of the regulation of CXCR4 in human breast cancer cells. Our results showed that CXCR4 expression was increased and PKCf activity involved in the HGF-mediated effects. We demonstrated that in breast cancer cells, PKCf was directly stimulated by HGF- activated signaling. Furthermore, blocking PKCf activity in MDA-MB-436 cells was found to significantly abrogate membrane CXCR4 expression. Importantly, inhibition of several other PKC isoenzymes did not produce any effect on MDA-MB-436 cells in terms of CXCR4 expression as well as migration, chemotaxis and invasion of the cells. In this study, the increase in CXCR4 expression in HGF- stimulated cells may be arising from enhanced transcription, but more importantly, from the prolonged time of membrane receptor exposure. Previous studies and our results on anti-CXCR4 Ab internalization suggested that HGF treatment led to a reduction in CXCR4 endocytosis. Consistent with published data, we also noted that HGF-activated CXCR4 expression was attenuated in the presence of PKCf inhibitor. The level of CXCR4 receptor expression may affect the strength of receptor signaling and determine the pattern of receptor-mediated effects. Protein phosphorylation is the most prevalent posttranslational modifica- tion and plays a major role in regulating protein function [49]. More importantly, as one of the earliest events in regulating G protein-coupled receptor (GPCR) signaling, phosphorylation initiates a process known as desensitization. Recent studies demonstrated that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases, which results in both positive and negative modulation of CXCR4 signaling [10]. The present study showed that CXCR4 phosphorylation was induced by HGF treatment in MDA-MB-436 or MCF-7 cells, and was attenuated by inhibition with PKCf. Therefore, we suggested that CXCR4 expression and CXCR4 phosphorylation correlate with PKCf in the HGF-mediated effects. Attempts to elucidate the molecular mechanisms underlying HGF-induced CXCR4 expression led to our interest in the role of Rac1, a member of the Ras superfamily of small guanosine triphosphatases (GTPases) that act as molecular switches to control cytoskeletal rearrangements and cell growth [50]. Indeed, the Rac family proteins, which are effectors of HGF signaling, are critical regulators of chemokine-induced integrin activation and motility [51]. Chianale and coworkers [23] have shown that in the presence of diacylglycerol kinase a, HGF promotes dissociation (from Rac/RhoGDI complex) and activation of Rac, which in turn promotes cytoplasm-to-membrane translocation and phos- phorylation of PKCf. Meanwhile, activated Rac-1 interacts with CXCR4 in lipid rafts on the cell surface resulting in enhanced sensitivity and responsiveness of hematopoietic cells to an SDF-1 gradient [52]. Other findings [53] indicated that binding of Rac1 to Part6 may lead to PKCf phosphorylation. Consistent with these studies, our results confirmed that: (1) HGF can promote Rac activation, which further promotes the cytoplasm-to-membrane translocation and phosphorylation of PKCf; (2) HGF stimulation contribute to longer duration for CXCR4 on cell membrane and reduce the CXCR4 endocytosis caused by CXCR4 antibodies. (3) NSC23766, a specific Rac1 inhibitor, can inhibit the activation of Rac1 and impair the HGF-induced PKCf phosphorylation; and (4) Blocking PKCf phosphorylation subsequently interfered with the membrane expression and phosphorylation of CXCR4. Therefore we hypothesized that HGF induces Rac recruitment at ruffling sites, which may prolong the membrane expression of CXCR4/Rac-1, and reduce the CXCR4 endocytosis caused by chemoattractants and/or CXCR4 antibodies, thereby stimulating the chemotaxis and migration of tumor cells. Subsequently, our findings suggested that functional Rac1 is required for HGF- induced PKCf activation. Rac1 and PKCf constitute a signaling pathway of HGF-induced CXCR4 expression. Thus far, a further elucidation is needed for the molecular mechanism that governs HGF-induced PKCf activation and CXCR4 expression. We know that HGF triggers motility dependently on the activation of several intracellular molecular pathways. The roles of PI 3-kinases [54,55], Ras and 41/43 kDa mitogen-activated protein kinase [56] in HGF-mediated cell scattering activity have also been established. PI 3K exists in a wide variety of cells and could be activated by most receptor tyrosine kinases, G protein-coupled receptors and non-receptor tyrosine kinase relative receptors. Akt is one of the downstream signals of the PI 3K pathway and is also a serine/threonine kinase. Recently, PI 3-kinase has been shown to mediate lymphocyte PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e29124