Dynamic relocalization of NHERF1 mediates chemotactic migration of ovarian cancer cells toward lysophosphatidic acid stimulation

NHERF1/EBP50 (Na⁺/H⁺ exchanger regulating factor 1; Ezrin-binding phosphoprotein of 50 kDa) organizes stable protein complexes beneath the apical membrane of polar epithelial cells. By contrast, in cancer cells without any fixed polarity, NHERF1 often localizes in the cytoplasm. The regulation of cytoplasmic NHERF1 and its role in cancer progression remain unclear. In this study, we found that, upon lysophosphatidic acid (LPA) stimulation, cytoplasmic NHERF1 rapidly translocated to the plasma membrane, and subsequently to cortical protrusion structures, of ovarian cancer cells. This movement depended on direct binding of NHERF1 to C-terminally phosphorylated ERM proteins (cpERMs). Moreover, NHERF1 depletion downregulated cpERMs and further impaired cpERM-dependent remodeling of the cell cortex, suggesting reciprocal regulation between these proteins. The LPA-induced protein complex was highly enriched in migratory pseudopodia, whose formation was impaired by overexpression of NHERF1 truncation mutants. Consistent with this, NHERF1 depletion in various types of cancer cells abolished chemotactic cell migration toward a LPA gradient. Taken together, our findings suggest that the high dynamics of cytosolic NHERF1 provide cancer cells with a means of controlling chemotactic migration. This capacity is likely to be essential for ovarian cancer progression in tumor microenvironments containing LPA.

Wnt5a stimulates chemotactic migration and chemokine production in human neutrophils

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (beta-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.

Phosphatidylinositol phosphates directly bind to neurofilament light chain (NF-L) for the regulation of NF-L self assembly

Phosphatidylinositol phosphates (PtdInsPs) are ubiquitous membrane phospholipids that play diverse roles in cell growth and differentiation. To clarify the regulation mechanism acting on neurofilament light chain (NF-L) self assembly, we examined the effects of various PtdInsPs on this process. We found that PtdInsPs, including PI(4,5)P2, directly bind to the positively charged Arg54 of murine NF-L, and this binding promotes NF-L self assembly in vitro. Mutant NF-L (R53A/R54A) proteins lacking binding affinity to PtdInsPs did not have the same effect, but the mutant NF-L proteins showed greater self assembly than the wild-type in the absence of any PtdInsP. These results collectively suggest that Arg54 plays a pivotal role in NF-L self assembly by binding with PtdInsPs.

A double point mutation in PCL-gamma1 (Y509A/F510A) enhances Y783 phosphorylation and inositol phospholipid-hydrolyzing activity upon EGF stimulation

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-gamma1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-gamma1, abolished interactions with translational elongation factor 1-alpha. Here, we report that the Y509A/F510A mutant PLC-gamma1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-gamma1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-gamma1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-gamma1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-gamma1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-gamma1 activation in vivo.

Pleckstrin homology domain of phospholipase C-gamma1 directly binds to 68-kDa neurofilament light chain

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.

Secretin induces neurite outgrowth of PC12 through cAMP-mitogen-activated protein kinase pathway

The gastrointestinal functions of secretin have been fairly well established. However, its function and mode of action within the nervous system remain largely unclear. To gain insight into this area, we have attempted to determine the effects of secretin on neuronal differentiation. Here, we report that secretin induces the generation of neurite outgrowth in pheochromocytoma PC12 cells. The expressions of Tau and beta-tubulin, neuronal differentiation markers, are increased upon secretin stimulation. In addition, secretin induces sustained mitogen-activated protein kinase (MAPK) activation and also stimulates the cAMP secretion. Moreover, the neurite outgrowth elicited by secretin is suppressed to a marked degree in the presence of either PD98059, a specific MAPK/ERK kinase (MEK) inhibitor, or H89, a specific protein kinase A (PKA) inhibitor. Taken together, these observations demonstrate that secretin induces neurite outgrowth of PC12 cells through cAMP-MAPK pathway, and provide a novel insight into the manner in which secretin participates in neuritogenesis.

Inositol 5'-phosphatase, SHIP1 interacts with phospholipase C-gamma1 and modulates EGF-induced PLC activity

Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.

Sphingosine mediates FTY720-induced apoptosis in LLC-PK1 cells

FTY720, a synthetic sphingoid base analog, was examined as a new sphingosine kinase inhibitor, which converts endogenous sphingosine into its phosphate form. With 20 micrometer of FTY720, sphingosine accumulated in the LLC-PK1 cells in a time- and dose-dependent manner. The FTY720 treated cells showed a high concentration of fragmented DNA, a high caspase-3 like activity and TUNEL staining cells. It was also found that the sphingosine and sphinganine level increased in a time- and dose-dependent manner within 12 h after the FTY720 treatment. The sphingosine kinase activity was reduced by FTY720 as much as other sphingosine kinase inhibitors, N, N-dimethylsphingosine (DMS), dl-threo-dihydrosphingosine (DHS). The fragmented DNA content as a result of the 20 micrometer of FTY720 treatment and by 5 micrometer of the exogenously added BSA-sphingosine complex indicated typical apoptosis. Under similar conditions, the accumulated sphingosine concentration in all the cells was almost identical even though the sphingosine distribution inside the cells was somewhat different. These results indicate that the FTY720 induced apoptosis is associated with the inhibition of the sphingosine kinase activity and is strongly associated with the successive accumulation of sphingosine.

Plasma cell granuloma in cyclosporin-induced gingival overgrowth: four cases report with interleukin-6 and phospholipase C-gammar1 immunohistochemistry

No abstract available.

Dexamethasone differentiates NG108-15 cells through cyclooxygenase1 induction

Cyclooxygenase (COX) is a key enzyme in the conversion of arachidonic acid into prostanoids which participate in various cellular functions including apoptosis, mitogenesis, inflammation, immune modulation and differentiation. Moreover, the synthetic glucocorticoid, dexamethasone has immune modulating and anti-inflammatory effects in vivo. Recently, dexamethasone was found to enhance retinoic acid-induced neuronal differentiation. In this study, we investigated the mechanisms of dexamethasone-mediated neuronal differentiation. Immunoblotting and morphological analysis demonstrated that dexamethasone induced neuronal differentiation through COX 1 induction. This phenomenon was inhibited by indomethacin, a COX inhibitor. In addition, the addition of exogenous prostaglandin E2 (PGE2), a substance produced by the COX-mediated pathway, triggered neurite outgrowth of cells treated with COX inhibitor. Taken together, COX 1 appears to play an important role in dexamethasone-mediated neuronal differentiation.

Plasma Cell Granuloma in Cyclosporine-Induced Gingival Overgrowth: A Report of Two Cases with Immunohistochemical Positivity of Interleukin-6 and Phospholipase C-gamma1

We report two cases of gingival plasma cell granuloma in a 34-yr-old and 40-yr-old two male renal transplant recipients with cyclosporine A (CsA)-induced gingival overgrowth (GO). Histologically, these lesions were composed of mature plasma cells, showing polyclonality for both lambda and kappa light chains and fibrovascular connective tissue stroma. In addition to the fact that CsA-induced plasma cell granuloma is rare, the salient features of our cases were the secretion of interleukin-6 and overexpression of phospholipase C-gamma1 of the tumor cells, which may explain the mechanisms of CsA- induced GO.

The synthetic peptide, His-Phe-Tyr-Leu-Pro-Met, is a chemoattractant for Jukat T cells

His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+]i) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanism.

The synthetic peptide, His-Phe-Tyr-Leu-Pro-Met, is a chemoattractant for Jukat T cells

His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+]i) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanism.

Immunohistochemical localization of eight phospholipase C isozymes in pancreatic islets of the mouse

The possible involvement of phospholipase C (PLC) in the regulation of insulin secretion is not clearly understood and neither its isozymes expressed nor cellular localization in the pancreatic islets is known. By using specific monoclonal antibodies, we have investigated the expression and localization of eight different PLC isozymes, beta1, beta2, beta3, beta4, gamma1, gamma2, delta1, and delta2, in the pancreatic islets of adult mice. Immunohistochemical analysis carried out on paraffin embedded sections showed a distinct pattern of expression for each of the PLC isozymes. In the central part of the islets containing beta cells, a high level of beta4 and moderate levels of beta3 and gamma1 were expressed, whereas PLC-beta1 and -gamma1 were abundantly expressed in the exocrine pancreas. These results demonstrated the heterogeneity in expression of the phospholipase C isozymes in pancreatic islets. It is conceivable that these isozymes are coupled to different receptors and perform selective tasks in the regulation of insulin secretion for glucose homeostasis.

Cloning and characterization of 5'-upstream region of human phospholipase C-beta2 gene

5'-upstream region of the phospholipase C-beta2 gene, 810 bp, was cloned and characterized. S1 nuclease mapping and primer extension analyses revealed that a single transcriptional start site locates at 284 nucleotides upstream from the beginning of translation. The 5-upstream region lacks both TATA motif and typical initiator sequence, but retains GC-rich segment. Two putative regulatory regions, a negative region (-636/-588) and a positive region (-98/ -13) were identified in the upstream region of PLC-beta2 gene. We suggest that the transcription of PLC-beta2 may be regulated by binding of regulatory proteins to the negative and/or positive regulatory regions located in the upstream of the gene.

Expression of Phospholipase C-gamma1 and gamma2 in Non-Hodgkin's and Hodgkin's Lymphoma

Phospholipase C (PLC) plays a role in ligand-mediated signal transduction for cellular activity such as proliferation and differentiation. A recent observation that PLC- gamma1 is highly expressed in some kinds of human cancer tissue supports the view that PLC-gamma1 may be involved in proliferation and carcinogenesis. PLC-gamma2 is known to be involved in B cell differentiation and maturation. However, there have been few studies about the expressions of PLC-gamma1 and gamma2 in human lymphoid malignancy. In the present study, we examined the contents of PLC-gamma1 and gamma2 in 10 cases of B cell, 10 cases of T cell non-Hodgkin's lymphoma and 5 cases of Hodgkin's lymphoma to find out whether these enzymes play any role in the carcinogenesis by immunohistochemistry and immunoprecipitation. Immunoprecipitation analysis revealed that in contrast to increased expression of PLC-gamma2 only in B cell lymphoma, a considerably higher level of PLC-gamma1 was detected in both B and T cell lymphoma. Immunohistochemical finding confirmed this observation. PLC-gamma1 and PLC-gamma2 were expressed in the cytoplasm of most tumor cells. PLC-gamma2 was also expressed in mature B cells, while PLC-gamma1 was not expressed in reactive non-tumor cells. These results suggest that PLC-gamma1 mediated signal transduction implicates a significant role in the carcinogenesis of all types of lymphoid tissue, and PLC-gamma2 may play a role in the carcinogenesis of B cell lymphoma as well as B cell differentiation.

The mechanism of phospholipase C-gamma1 regulation

Phospholipase C (PLC)1 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces a transient increase in intracellular free Ca2+, while DAG directly activates protein kinase C. Upon stimulation of cells with growth factors, PLC-gamma1 is activated upon their association with and phosphorylation by receptor and non-receptor tyrosine kinases. In this review, we will focus on the activation mechanism and regulatory function of PLC-gamma1.

Mass Production and Characterization of Anti-HBsAg Human Antibody B7 Fd

We expressed anti-HBsAg human antibody fragment (B7 Fd) using pRSET-A vector and BL21(DE3)pLysS strain of E. coli. B7 Fd is composed only of variable domain (VH) and CH1 constant domain of IgG heavy chain molecule. This Fd molecule was solubilized using guanidine salt and then expressed in the form of inclusion body and successfully refolded into functional antibody molecule by rapid dilution in refolding buffer. B7 Fd reacted with d epitope of hepatitis B virus surface antigen (HBsAg). Its affinity was determined by competition enzyme-linked immunosorbent assay (competition ELISA). The K value of B7 Fd was 3.3 * 10.

The mRNA Expression of Phosphoinositide-specific Phospholipase C-beta Isozymes in Postnatal Rat Brains / 대한해부학회지

Phosphoinositide-specific phospholipase C(PLC) is known as a key enzyme which produces two major second messengers: diacylglycerol and inositol 1,4,5 trisphosphate. Although it has been suggested that PLC beta isozymes have important roles in nervous system, less is known about the function of PLC beta in development of nervous system. We have localized the mRNA expressions of PLC beta isozymes in the postnatal rat brains by id firm hybridization histochemistry. In the postnatal rat brains, each isozyme of PLC beta showed differential expression pattern. The expression of PLC beta1 mRNA was found in various areas including olfactory bulb, cerebral cortex, caudate putamen, hippocampus, dentate gyrus, and cerebellum. In general, the expression in these areas was gradually increased after birth (PO) until postnatal day 21 (P2l) and slightly decreased to adult level. The expression of PLC beta2 mRNA was not found in postnatal rat brains. The expression of PLC beta3 mRNA was found from P0, peaked at Pl4, and decreased to adult level in the purkinje cells of cerebellum. PLC beta4 mRNA was strongly expressed in the thalamus, cerebellum, cerebral cortex, and olfactory bulb. In these areas, the expression was gradually increased after birth, peaked at P2l, and decreased to adult level. In whole body parasagittal sections of 18 day old rat embryo, PLC betal mRNA was exclusively expressed in nervous tissue, PLC beta3 and PLC beta4 were expressed in various tissues, and the expression of PLC beta2 was not found in any kind of rat tissues. From the different spatiotemporal mRNA expression patterns of PLC beta isozymes in the postnatal rat brains, it is suspected that each PLC beta isozyme may have specific role in signal transduction for postnatal development of rat brain.

Expression, Characterization and Chain Shuffling of an Anti-HBsAg Phage Antibody

In our previous report an anti-HBsAg human monoclonal antibody was generated using antibody phage display library technique. Using pComb3 filamentous phagemid vector, Fab molecule was expressed in fusion form to a phage coat protein in the periplasm of E. coli. A clone of HBsAg binder was selected after panning and designated as B7. In order to select the clones with higher affinity and to examine which chain contributes most to the affinity of B7, the light and heavy chain of B7 was sequentially deleted and replaced with new library. HBsAg-binders were selected and tested by EIA (enzyme immunoassay). It was revealed that the affinity of B7 depends only on the heavy chain of Fd. B7 Fd was constructed without light chain and specificity and affinity was further confirmed by western blot analysis. This human monoclonal Fd antibody was found to react with d antigenic determinant of HBsAg as the original clone did. The nucleotide sequence analysis revealed that VH of B7 could be classified into Kabat's subgroup II and human IgG heavy chain family IV. The CH1 of B7 was IgG1.