Tzeng Horng Leu

A Role for the PI-3 Kinase Signaling Pathway in Fear Conditioning and Synaptic Plasticity in the Amygdala

Western blot analysis of neuronal tissues taken from fear-conditioned rats showed a selective activation of phosphatidylinositol 3-kinase (PI-3 kinase) in the amygdala. PI-3 kinase was also activated in response to long-term potentiation (LTP)-inducing tetanic stimulation. PI-3 kinase inhibitors blocked tetanus-induced LTP as well as PI-3 kinase activation. In parallel, these inhibitors interfered with long-term fear memory while leaving short-term memory intact. Tetanus and forskolin-induced activation of mitogen-activated protein kinase (MAPK) was blocked by PI-3 kinase inhibitors, which also inhibited cAMP response element binding protein (CREB) phosphorylation. These results provide novel evidence of a requirement of PI-3 kinase activation in the amygdala for synaptic plasticity and memory consolidation, and this activation may occur at a point upstream of MAPK activation.

Functional implication of the interaction between EGF receptor and c-Src.

EGF receptor (EGFR) and c-Src are the prototypes that individually represent the receptor and nonreceptor tyrosine kinases respectively. Accumulated evidence reveals the association between EGF-activated EGFR and c-Src that leads to activation of both kinases. Importantly, their mutual interaction is required for many EGFR-mediated cellular functions including proliferation, migration, survival and EGFR endocytosis. Interestingly, activation of c-Src and its association with transactivated EGFR is also observed in cells stimulated with non-EGF agonists. This review will not only discuss the structure, function and regulation of these two tyrosine kinases, but also will summarize our current knowledge of the molecular mechanisms depicting the cellular events that require their participation.

Src oncogene activates MMP-2 expression via the ERK/Sp1 pathway

Previous studies demonstrated that activation of Src oncogene increased matrix metalloproteinase‐2 (MMP‐2) expression in various types of cells. In this study, we elucidated the underlying mechanism of Src‐induced MMP‐2. We first used murine fibroblast cell line C3H10T1/2 and its v‐Src transfectant IV5 to address this issue. RT‐PCR and promoter activity assay indicated that Src stimulated MMP‐2 via transcriptional activation. Transfection of constitutively active Src into C3H10T1/2 cells also stimulated MMP‐2 mRNA expression. Deletion and mutation analysis indicated that the Sp1 site located within the −91/−84 region of human MMP‐2 promoter is the major responsive element for Src. Electrophoresis mobility shift assays showed that Src enhanced the binding of Sp1 to this consensus site to stimulate MMP‐2 gene expression. We next investigated the signaling pathway that mediated the effect of Src on MMP‐2. Our data showed that extracellular signal‐regulated kinase (ERK) pathway inhibitor PD98059, but not the c‐Jun N‐terminal kinase (JNK) inhibitor SP600125, p38 kinase inhibitor SB203580, and PI3K inhibitor wortmannin, attenuated Src‐induced MMP‐2 promoter activity. These inhibitors did not show significant effect on basal MMP‐2 promoter activity in C3H10T1/2 cells. In addition, the dominant negative mutant of ERK‐2 suppressed the activation of MMP‐2 by Src. Treatment of PD98059 or an Src specific inhibitor PP1 reduced Sp1 DNA binding activity in IV5 cells. Taken together, our results strongly suggest that Src induces MMP‐2 expression via transcription activation and the ERK/Sp1 signaling pathway is involved in this process. J. Cell. Physiol.

Inhibition of cell migration by autophosphorylated mammalian sterile 20-like kinase 3 (MST3) involves paxillin and protein-tyrosine phosphatase-PEST.

MST3 is a member of the sterile-20 protein kinase family with a unique preference for manganese ion as a cofactor in vitro; however, its biological function is largely unknown. Suppression of endogenous MST3 by small interference RNA enhanced cellular migration in MCF-7 cells with reduced expression of E-cadherin at the edge of migrating cells. The alteration of cellular migration and protruding can be rescued by RNA interference-resistant MST3. The expression of surface integrin and Golgi apparatus was not altered, but phosphorylation on tyrosine 118 and tyrosine 31 of paxillin was attenuated by MST3 small interfering RNA (siRNA). Threonine 178 was determined to be one of the two main autophosphorylation sites of MST3 in vitro. Mutant T178A MST3, containing alanine instead of threonine at codon 178, lost autophosphorylation and kinase activities. Overexpression of wild type MST3, but not the T178A mutant MST3, inhibited migration and spreading in Madin-Darby canine kidney cells. MST3 could phosphorylate the protein-tyrosine phosphatase (PTP)-PEST and inhibit the tyrosine phosphatase activity of PTP-PEST. We conclude that MST3 inhibits cell migration in a fashion dependent on autophosphorylation and may regulate paxillin phosphorylation through tyrosine phosphatase PTP-PEST.

Overexpression of p97Eps8 leads to cellular transformatioan: Implication of pleckstrin homology domain in p97Eps8-mediated ERK activation

Two isoforms of Eps8, p97Eps8 and p68Eps8, have been identified as the substrates for receptor tyrosine kinases. Our previous studies indicated that both tyrosyl phosphorylation and protein expression of Eps8 were elevated in v-Src transformed cells. In an attempt to examine the role played by p97Eps8 in tumorigenesis, we have first obtained cells overexpressing p97Eps8 and its pleckstrin homology (PH)-truncated variant. We then demonstrated that cells overexpressing p97Eps8 not only exhibited the ability of focus formation in cell culture but also promoted the tumor formation in mice as compared to controls. Furthermore, elevated serum-induced extracellular responsive kinase (ERK) activation was observed in p97Eps8 overexpressors. This enhanced ERK activation was sensitive to a MEK1 specific inhibitor PD98059 and was important for p97Eps8-mediated transformation, since transfection of vectors expressing dominant negative MEK1 and p97Eps8 abrogated focus formation by p97Eps8. In contrast, PH-truncated p97Eps8 failed to localize at the plasma membrane and that the truncated variant also did not elevate ERK activation and cellular transformation in response to serum stimulation. Our results thus indicated that: (i) the gene encoding p97Eps8 was an oncogene; (ii) p97Eps8-induced oncogenesis was partly mediated by ERK activation; and (iii) the PH domain of p97Eps8 was critical for its cellular localization, ERK activation and its ability to transform cells.

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.

Lipopolysaccharide-promoted proliferation of Caco-2 cells is mediated by c-Src induction and ERK activation

As a major component of the cell wall of Gram-negative bacteria, lipopolysaccharide (LPS) can be released into the bloodstream to cause a spectrum of pathophysiological reactions. Despite the fact that colon epithelium cells in situ are continuously exposed to LPS, their biological responses as provoked by LPS as well as the underlying mechanisms are poorly defined. In the present study, we observed that LPS directly stimulated growth of Caco-2 cells as well as enhanced the amounts of c-Src, which could be partly attributable to increased c-src transcript. Parallel to LPS-induced c-Src expression was FAK activation and ERK activation. Remarkably, activation of ERK and cellular proliferation by LPS could be inhibited by PP2, the specific Src inhibitor, implicating the essential role of c-Src in this process. To our knowledge, this is the first report indicating that LPS can increase cellular growth via upregulation of c-Src in colon epithelial cells.

Translational up-regulation of Aurora-A in EGFR-overexpressed cancer.

Abnormal expression of Aurora‐A and epidermal growth factor receptor (EGFR) is observed in different kinds of cancer and associated with poor prognosis in cancer patients. However, the relationship between Aurora‐A and EGFR in tumour development was not clear. In previous reports, we found that EGFR translocates to nucleus to activate Aurora‐A expression after EGF treatment in EGFR‐overexpressed cells. However, we also observed that not all the EGFR‐overexpressed cells have the nuclear EGFR pathway to mediate the Aurora‐A expression. In this study, we demonstrated that EGF signalling increased the Aurora‐A protein expression in EGFR‐overexpressed colorectal cancer cell lines via increasing the translational efficiency. In addition, the overexpression of EGFR was also associated with higher expression of Aurora‐A in clinical colorectal samples. Activation of the PI3K/Akt/mTOR and MEK/ERK pathways mediated the effect of EGF‐induced translational up‐regulation. Besides, only the splicing variants containing exon 2 of Aurora‐A mRNA showed increased interaction with the translational complex to synthesize Aurora‐A protein under EGF stimulus. Besides, the exon 2 containing splicing variants were the major Aurora‐A splicing forms expressed in human colorectal cancers. Taken together, our results propose a novel regulatory mechanism for the abnormal expression of Aurora‐A in EGFR‐overexpressed cancers, and highlight the importance of alternative 5′‐UTR splicing variants in regulating Aurora‐A expression. Furthermore, the specific expression of exon 2 containing splicing variants in cancer tissues may serve as a potential target for cancer therapy in the future.