HGF induced CXCR4 expression in a PKCf-dependent fashion To investigate the mechanisms underlying the effects of HGF on CXCR4 expression we examined downstream constituents of the HGF-activated pathway in MDA-MB-436 cells. Studies have shown that cell responses to HGF/SF binding involve activation of different functional PKC subspecies that are associated with enhanced cell migration and growth [14,29]. We thus focused on PKCf which had recently been implicated in HGF-induced signaling [23]. Activation of PKC requires phosphorylation by an upstream kinase which results in translocation to the plasma membrane where PKC can phosphorylate its substrate. To verify whether HGF/SF activates conventional or novel PKC isoforms in MDA- MB-436 cells, we performed western blot analyses of extracts from treated and unstimulated cells, and found that HGF had a major effect on PKCf activity. As shown in Figure 3A, the level of phosphorylated PKCf in MDA-MB-436 and MCF-7 cells increased by approximately 3- to 10-fold within 5 to 60 minutes after HGF treatment, in consistency with a hallmark feature of PKCf activation (Figure 3A, Figure S2A). To obtain an appreciable down-regulation of cellular atypical PKCs, we silenced PKCf using different combinations of specific siRNAs. This resulted in impairment of HGF-induced expression and phosphorylation of both PKCf and CXCR4 (Figures 3B–3C, Figures S2B–2C). Addition of the PKCf inhibitory pseudosub- strate (PSf) was shown to substantially affect the basal CXCR4 expression and completely abrogated HGF-induced CXCR4 expression in MDA-MB-436 cells (Figures 3D–3E). In contrast, inhibition of other PKC isoenzymes (PKCe and PKCa/b) did not produce any change in CXCR4 expression and phosphorylation (Figure 3D, Figures S2D–2E)). Moreover, after treatment with cholesterol sulfate (a chemical activator of PKCf [31]), the expression of membrane CXCR4 proteins in MDA-MB-436 cells was significantly enhanced (Figure 3E), which was similar to that in HGF-treated cells. Members of the Rac small GTPases family have been implicated in the spread and dissociation of the HGF-induced activation of the Cdc42/Rac-regulated p21-activated kinase (PAK) and c-Jun N-terminal kinase to membrane ruffles [32]. An increasing body of evidence indicates that Rac mediates the localization and activation of atypical PKCf in epithelial cells [23,33]. Therefore, we examined the involvement of Rac-GTPases in HGF-induced PKCf activation, and found that treatment with HGF increased Rac1 activity in MDA-MB-436 and MCF-7 cells (Figure 3F, Figure S2F). For an insight into this observation, our test with NSC23766 (a Rac-specific small-molecule inhibitor that targets Rac activation by GEF [34]) demonstrated that the HGF- induced increase in Rac1 activities can be blocked by Rac1 inhibitor, and that the activities of Rac1 was involved in HGF- induced PKCf phosphorylation. The concentration of Rac1 inhibitor was inversely associated with the level of HGF-induced PKCf phosphorylation (Figure 3G, Figure S2G). To directly test the role of total Rac activation levels in regulating HGF-induced PKCf phosphorylation in breast cancer cells, levels of Rac1 were knocked down using RNA interference with small interfering (si)RNA. Reductions in Rac protein levels resulted in proportional changes in Rac activity, and in turn, interfered with PKCf phosphorylation and expression of CXCR4 (Figure 3H, Figure S2H). Immunocytochemical analysis revealed that NSC23766 completely abrogated the HGF-induced translocation of PKCf from cytoplasm to cell membrane in MDA-MB-436 cells (Figure 3I), suggesting that functional Rac1 might be required for PKCf activation by HGF. Importantly, blocking Rac1 PKCf Induced by HGF Increases CXCR4 Expression activation in MDA-MB-436 cells caused a reduction in membra- nal and intracellular CXCR4 expression comparable to that of PSf or PKCf siRNAs in a manner that was neither synergistic nor additive in the presence of the both inhibitors (Figure 3C–3D, Figure 3I). This result supported our hypothesis that Rac1 and PKCf are involved in the same pathway in HGF-stimulated elevation of CXCR4 expression. HGF-induced CXCR4 expression was functional and depended on PKCf for its activity The role of CXCR4 in cancer depends on whether it is in an activated signaling state. CXCR4 is phosphorylated in response to binding of CXCL12 in a G protein-coupled receptor kinase 2- dependent fashion [35]. Receptor phosphorylation stimulates the interaction between b-arrestin and the carboxy terminus [36]. Phosphorylation of CXCR4 can also occur in response to the activation of other receptors and involve additional kinases such as protein kinase C [37] or tyrosine kinases [12]. To better define the significance of HGF-induced CXCR4 expression, we designed the following experiments. The first set of experiments was based on western blot analysis of MDA-MB-436 cells treated with HGF or SDF-1. The level of phosphorylated CXCR4 was dramatically increased after treatment with either HGF or SDF-1 (Figure 2B, Figure S1A). Cholesterol sulfate increased the level of phosphor- ylated CXCR4 similarly to as found in HGF-treated cells. Addition of PKCf inhibitory PSf was found to substantially affect the expression of phosphorylated CXCR4. In contrast, inhibition of other PKC isoenzymes (PKCe and PKCa/b) produced no change in expression of phosphorylated CXCR4 (Figure 3D, Figure S2E). To better understand the steps in which CXCR4 may contribute to HGF-induced invasion, we examined the migration and chemotatic activities of MDA-MB-436 cells in response to HGF stimulation. As shown by the wound healing assays, the migration of HGF-stimulated cell was more efficient compared with PBS-treated cells (Figures 4A–4B), occurring in a dose- dependent manner (Figure 4C). In the presence of 10 mM PSf or 1 mM AMD3100 (a small-molecule inhibitor of CXCR4), HGF- induced migration was inhibited, which further supported our hypothesis that HGF-stimulated MDA-MB-436 cell migration is mediated by the CXCR4/PKCf pathway (Figure 4B). We then examined the chemotactic effects of CXCR4 using HGF and SDF-1 gradients. SDF-1 was found to induce the chemotaxis of MDA-MB-436 cells in a dose-dependent manner. HGF-induced chemotaxis also exhibited a typical bell-shaped dose-response curve, and the chemotaxis indexes were 1.5-fold greater than those with SDF-1 induction (Figure 4D–4E). Adding PSf or cheler- ythrine chloride (an inhibitor of all PKCs) substantially affected and even abrogated HGF-induced chemotaxis in MDA-MB-436 cells. In contrast, PKCe and PKCa/b, the other PKC isoenzyme inhibitors, had no effect on HGF-induced chemotaxis (Figure 4E). Given that HGF induces CXCR4 expression, and that HGF as well as CXCR4/SDF-1 interaction plays an important role in metastasis, we set to examine whether the SDF-1/CXCR4 interaction contributes to HGF-induced invasive activities. Inva- sion assays were performed using transwell invasion chambers. When 50 ng/ml of HGF was added to the supplemented medium, there was a 3- to 5-fold increase in the number of invading cells as compared with the PBS-treated cells. These results indicated that HGF enhanced the invasive activity of MDA-MB-436 cells toward SDF-1 in vitro (Figure 4F, Figure S3A–3B). The results showed that HGF did not influence cancer cell viability and proliferation (Figure S3C–3D). These data were consistent with previous report PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e29124