Yen Jen Chen

EPS8 Facilitates Cellular Growth and Motility of Colon Cancer Cells by Increasing the Expression and Activity of Focal Adhesion Kinase

In an attempt to study the role of Eps8 in human carcinogenesis, we observe that ectopic overexpression of Eps8 in SW480 cells (low Eps8 expression) increases cell proliferation. By contrast, expressing eps8 small interference RNA in SW620 and WiDr cells (high Eps8 expression) reduces their proliferation rate. Interestingly, attenuation of Eps8 decreases Src Pi-Tyr-416, Shc Pi-Tyr-317, and serum-induced FAK Pi-Tyr-397 and Pi-Tyr-861. Remarkably, by virtue of mammalian target of rapamycin/STAT3 Pi-Ser-727, Eps8 modulates FAK expression required for cell proliferation. Within 62% of colorectal tumor specimens examined, >2-fold enhancement of Eps8 as compared with their normal counterparts is observed, especially for those from the advanced stage. In agreement with the modulation of FAK by Eps8, the concomitant expression of these two proteins in tumor specimens is observed. Notably, Eps8 attenuation also impedes the motility of SW620 and WiDr cells, which can be rescued by ectopically expressed FAK. This finding discloses the indispensability of Eps8 and FAK in cell locomotion. These results provide a novel mechanism for Eps8-mediated FAK expression and activation in colon cancer cells.

Requirement of Inducible Nitric-oxide Synthase in Lipopolysaccharide-mediated Src Induction and Macrophage Migration

Previously, we have demonstrated the induction of Src in lipopolysaccharide (LPS)-stimulated macrophages. In this study, we observed that pharmacological blockade or knockout of inducible nitric-oxide synthase (iNOS) reduced LPS-mediated Src induction and macrophage migration. Either SNAP (a NO donor) or 8-Br-cGMP (a cGMP analogue) could rescue these defects in iNOS-null macrophages, which indicated the participation of NO/cGMP in LPS-elicited Src expression and mobilization. In addition, Src family kinase (SFK)-specific inhibitor, PP2, inhibited SNAP- and 8-Br-cGMP-evoked motility implicating the involvement of SFKs downstream of NO/cGMP. Analysis of the expression of SFKs indicated LPS dramatically induced Src, which could be attributable to the increased level of the src transcript. Attenuation of Src by src-specific siRNA reduced LPS- and SNAP-evoked mobilization in Raw264.7 macrophages, and reintroduction of avian Src could rescue their motility. Furthermore, LPS-mediated Src induction led to increased FAK Pi-Tyr-397 and Pi-Tyr-861, which was also iNOS-dependent. With these findings, we concluded that iNOS was important for LPS-mediated macrophage locomotion and Src was a critical player in this process.

Eps8 Protein Facilitates Phagocytosis by Increasing TLR4-MyD88 Protein Interaction in Lipopolysaccharide-stimulated Macrophages

Background: TLR4-mediated p38 MAPK and actin cytoskeleton reorganization are important for macrophage phagocytosis. Results: TLR4-induced Eps8 facilitates TLR4-MyD88 interaction, leading to the activation of p38 MAPK, actin polymerization, and the uptake of bacteria in macrophages. Conclusion: Eps8 is critical in innate immunity. Significance: This is the first report to indicate that Eps8 contributes to efficient phagocytosis in macrophages. Toll-like receptors (TLRs) are crucial in macrophage phagocytosis, which is pivotal in host innate immune response. However, the detailed mechanism is not fully defined. Here, we demonstrated that the induction of Src and Eps8 in LPS-treated macrophages was TLR4- and MyD88-dependent, and their attenuation reduced LPS-promoted phagocytosis. Confocal microscopy indicated the colocalization of Eps8 and TLR4 in the cytosol and at the phagosome. Consistently, both Eps8 and TLR4 were present in the same immunocomplex regardless of LPS stimulation. Inhibition of this complex formation by eps8 siRNA or overexpression of pleckstrin homology domain-truncated Eps8 (i.e. 261-p97Eps8) decreased LPS-induced TLR4-MyD88 interaction and the following activation of Src, focal adhesion kinase, and p38 MAPK. Importantly, attenuation of Eps8 impaired the bacterium-killing ability of macrophages. Thus, Eps8 is a key regulator of the LPS-stimulated TLR4-MyD88 interaction and contributes to macrophage phagocytosis.

The iNOS/Src/FAK axis is critical in Toll-like receptor-mediated cell motility in macrophages

The Toll-like receptors (TLRs) play a pivotal role in innate immunity for the detection of highly conserved, pathogen-expressed molecules. Previously, we demonstrated that lipopolysaccharide (LPS, TLR4 ligand)-increased macrophage motility required the participation of Src and FAK, which was inducible nitric oxide synthase (iNOS)-dependent. To investigate whether this iNOS/Src/FAK pathway is a general mechanism for macrophages to mobilize in response to engagement of TLRs other than TLR4, peptidoglycan (PGN, TLR2 ligand), polyinosinic-polycytidylic acid (polyI:C, TLR3 ligand) and CpG-oligodeoxynucleotides (CpG, TLR9 ligand) were used to treat macrophages in this study. Like LPS stimulation, simultaneous increase of cell motility and Src (but not Fgr, Hck, and Lyn) was detected in RAW264.7, peritoneal macrophages, and bone marrow-derived macrophages exposed to PGN, polyI:C and CpG. Attenuation of Src suppressed PGN-, polyI:C-, and CpG-elicited movement and the level of FAK Pi-Tyr861, which could be reversed by the reintroduction of siRNA-resistant Src. Besides, knockdown of FAK reduced the mobility of macrophages stimulated with anyone of these TLR ligands. Remarkably, PGN-, polyI:C-, and CpG-induced Src expression, FAK Pi-Tyr861, and cell mobility were inhibited in macrophages devoid of iNOS, indicating the importance of iNOS. These findings corroborate that iNOS/Src/FAK axis occupies a central role in macrophage locomotion in response to engagement of TLRs.

Butyrate regulates the expression of c-Src and focal adhesion kinase and inhibits cell invasion of human colon cancer cells.

Epidemiological studies indicate that dietary fiber‐derived fermentation products such as butyrate can prevent colon cancer development. To further dissect the role of butyrate in anticarcinogenesis, its effect on cellular growth and invasion as well as the expression of c‐Src and FAK, two mutually interactive nonreceptor tyrosine kinases, in three different human colon cancer cell lines (Caco‐2, SW480, and SW620) were investigated. In addition to growth inhibition, butyrate treatment results in a significant downregulation of c‐Src and FAK in human colon cancer cells, which can be attributable to their reduced transcripts and implicates the participation of a butyrate‐sensitive pathway in modulating their expression. Concurrent to butyrate‐reduced c‐Src and FAK expression is the decrease of FAK Tyr‐decrease 397 phosphorylation. Besides, butyrate also abolished the secretion of MMP‐2 and MMP‐9. And these butyrate‐mediated effects severely impaired invasion of SW620 cells through Matrigel in vitro. Interestingly, in situ parallel enhancement of c‐Src and FAK was also observed in human colorectal tumor specimens. These results imply that by virtue of suppression of c‐Src and FAK along with other butyrate targets in colonocytes, butyrate could effectively inhibit tumor growth and invasion.

The Inducible Nitric-oxide Synthase (iNOS)/Src Axis Mediates Toll-like Receptor 3 Tyrosine 759 Phosphorylation and Enhances Its Signal Transduction, Leading to Interferon-β Synthesis in Macrophages

Background: TLR3 Tyr-759 phosphorylation is critical in dsRNA-mediated IFN-β production. Results: iNOS-induced Src expression and activation amplify IFN-β production through induction of TLR3 Tyr-759 phosphorylation in macrophages. Conclusion: Src-TLR3 interaction is important in dsRNA- and LPS-induced IFN-β production. Significance: An essential role of the iNOS/Src/TLR3 axis is established in IFN-β production in macrophages that might have clinical applications for infectious diseases. Double-stranded RNA (dsRNA) induces phosphorylation of Toll-like receptor 3 (TLR3) at tyrosine 759 and subsequently triggers signaling pathways to promote interferon-β (IFN-β) production. In this study, we found that dsRNA stimulation induces biphasic TLR3 Tyr-759 phosphorylation in macrophages. In addition to the immediate TLR3 Tyr-759 phosphorylation, we identified a second wave of Tyr-759 phosphorylation accompanied by an increase of both Src and ifn-β transcription in the later phase of dsRNA stimulation. Interestingly, Src phosphorylated TLR3 Tyr-759 in vitro and in vivo. However, knockdown of Src abolished the late phase of TLR3 Tyr-759 phosphorylation and decreased the nuclear accumulation of interferon regulatory factors 3 and 7 (IRF3 and -7) and IFN-β production. Reintroduction of Src restored all of these molecular changes. Notably, via down-regulation of Src, dsRNA-elicited TLR3 Tyr-759 phosphorylation, the nuclear accumulation of IRF3/IRF7, and IFN-β generation were inhibited in inducible nitric-oxide synthase (iNOS)-null macrophages. TLR3 knockdown destabilized Src and reduced the nuclear level of IRF3/IRF7 and IFN-β production in macrophages exposed to LPS (a TLR4 ligand known to induce Src and IFN-β expression). Ectopic expression of wild type TLR3, but not its 759-phenylalanine mutant, restored Src activity and ifn-β transcription. Taken together, these results suggested an essential role of the iNOS/Src/TLR3 axis in IFN-β production in macrophages.

Berberine reduces Toll-like receptor-mediated macrophage migration by suppression of Src enhancement.

Berberine is an isoquinoline with anti-inflammatory activity. We previously demonstrated that there was a loop of signal amplification between nuclear factor kappa B and Src for macrophage mobility triggered by the engagement of Toll-like receptors (TLRs). The simultaneous suppression of lipopolysaccharide (LPS)-mediated upregulation of inducible nitric oxide synthase, cyclooxygenase 2, and cell mobility in berberine-treated macrophages suggested Src might be a target of berberine. Indeed, th reduced migration, greatly suppressed Src induction in both protein and RNA transcript by berberine were observed in macrophages exposed to LPS, peptidoglycan, polyinosinic-polycytidylic acid, and CpG-oligodeoxynucleotides. In addition to Src induction, berberine also inhibited LPS-mediated Src activation in Src overexpressing macrophages and S-nitroso-N-acetylpenicillamine (a nitric oxide donor) could partly restore it. Moreover, berberine suppressed Src activity in fibronectin-stimulated macrophages and in v-Src transformed cells. These results implied that by effectively reducing Src expression and activity, berberine inhibited TLR-mediated cell motility in macrophages.