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 PKCζ activity involved in the HGF-mediated effects. We demonstrated that in breast cancer cells, PKCζ was directly stimulated by HGF-activated signaling. Furthermore, blocking PKCζ 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 PKCζ 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 modification and plays a major role in regulating protein function 
. 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 
. 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 PKCζ. Therefore, we suggested that CXCR4 expression and CXCR4 phosphorylation correlate with PKCζ 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 
. Indeed, the Rac family proteins, which are effectors of HGF signaling, are critical regulators of chemokine-induced integrin activation and motility 
. Chianale and coworkers 
have shown that in the presence of diacylglycerol kinase α, HGF promotes dissociation (from Rac/RhoGDI complex) and activation of Rac, which in turn promotes cytoplasm-to-membrane translocation and phosphorylation of PKCζ. 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 
. Other findings 
indicated that binding of Rac1 to Part6 may lead to PKCζ 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 PKCζ; (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 PKCζ phosphorylation; and (4) Blocking PKCζ 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 PKCζ activation. Rac1 and PKCζ constitute a signaling pathway of HGF-induced CXCR4 expression.
Thus far, a further elucidation is needed for the molecular mechanism that governs HGF-induced PKCζ 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 
, Ras and 41/43 kDa mitogen-activated protein kinase 
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 adhesion and migration by regulating Rho and PKCζ 
. In order to investigate whether PI3K/Akt were involved in the effects of HGF-induced CXCR4 expression in MDA-MB 436 cells, we examined the changes of CXCR4 expression after HGF-induced PKCζ activation by PI3K/Akt inhibitors. Western blot assays and immunofluorescent staining data suggested that PI 3K/Akt are required for PKCζ activation and CXCR4 expression. Furthermore, Rac1 activation is also dependent on PI3-kinase activity. The lack of complete inhibition may indicate that activation of a small percentage of Akt/PKB is sufficient for chemotaxis, or that an unidentified signaling molecule downstream of the PI 3K shares a partially redundant function with Akt/PKB. Those results also indicated that the mechanism of HGF-induced CXCR4 expression in the MDA-MB-436 breast cancer cell line involved a pathway of activation of the PKCζ and PI 3K/Akt, and that signals stimulated by HGF passed on from PI 3K/Akt to Rac1 and then to PKCζ. These were further supported by the fact that multiple chemical inhibitors and PSζ can block signal transmission.
MT1-MMP, also known as MMP-14, has a single transmembrane domain and is an integral membrane protein with an extracellular catalytic domain suggested to be a key enzyme in tumor metastasis and angiogenesis. Numerous studies implied that MT1-MMP in breast tumors are correlated with the outcome of patients with breast cancer 
. Reduction of MT1-MMP from the breast cancer cells resulted in significant reduction of in vitro invasiveness and loss of response to an invasion stimulus. An association between MT1-MMP and the PKCζ activation has been reported. PKCζ may be responsible for the increased binding of Sp1 to MT1-MMP promoter site in shear-stressed endothelial cell (EC) and thus the induction of MT1-MMP protein 
. MT1-MMP in coordination with CXCR4 promotes invasion and dissemination of the tumor 
. HGF has also been shown to actively promote production of MT1-MMP in MDA-MB 231 cells, which further activated MMP2 and enhanced the invasiveness of breast cancer cells 
. Thus, we hypothesized that PKCζ may be involved in the signaling pathway that mediates MT1- MMP expression in cells exposed to HGF. Our data were consistent with the requirement of PKCζ phosphorylation and activation for Rac1 increased MT1-MMP expression by MDA-MB 436 cells. Inhibition of PKCζ, Rac-1 and phosphatidylinositol 3-kinase by their respective inhibitors PSζ, NSC23766 and LY290042 attenuated MT1-MMP expression in MDA-MB 436 cells. Therefore, the proper function of MT1-MMP may depend on both cellular expression and specific membrane localization. This study demonstrated that PKCζ is critical to regulation of MT1-MMP expression. Activation of PKCζ is also required in both EGFR- and chemokine receptor-mediated chemotaxis, suggesting the use of PKCζ as a promising target for more potent anti-metastasis therapeutic strategies. Because PKCζ-deficient mice are grossly normal, the pharmacologic inhibition of this enzyme should not be lethal to the hosts 
. The identification of PKCζ as a convergence point of EGFR- and chemokine receptor-mediated chemotaxis provided us with a potential novel target for anticancer drugs. We postulated that blocking PKCζ may completely impair the chemotactic activities of some cancer cells and result in reduced in tumor invasion and spreading.
Taken together, the results of our study provided new insights to molecular regulation of CXCR4 and to certain distinct regulatory molecules that can be targeted to modulate the CXCR4 signaling in breast cancer.