Zen Kouchi

Lipid Rafts and Caveolin-1 Are Required for Invadopodia Formation and Extracellular Matrix Degradation by Human Breast Cancer Cells

Invadopodia are ventral membrane protrusions through which invasive cancer cells degrade the extracellular matrix. They are thought to function in the migration of cancer cells through tissue barriers, which is necessary for cancer invasion and metastasis. Although many protein components of invadopodia have been identified, the organization and the role of membrane lipids in invadopodia are not well understood. In this study, the role of lipid rafts, which are cholesterol-enriched membrane microdomains, in the assembly and function of invadopodia in human breast cancer cells was investigated. Lipid rafts are enriched, internalized, and dynamically trafficked at invadopodia sites. Perturbation of lipid raft formation due to depleting or sequestering membrane cholesterol blocked the invadopodia-mediated degradation of the gelatin matrix. Caveolin-1 (Cav-1), a resident protein of lipid rafts and caveolae, accumulates at invadopodia and colocalizes with the internalized lipid raft membranes. Membrane type 1 matrix metalloproteinase (MT1-MMP), a matrix proteinase associated with invadopodia, is localized at lipid raft-enriched membrane fractions and cotrafficked and colocalized with Cav-1 at invadopodia. The small interfering RNA-mediated silencing of Cav-1 inhibited the invadopodia-mediated and MT1-MMP-dependent degradation of the gelatin matrix. Furthermore, Cav-1 and MT1-MMP are coexpressed in invasive human breast cancer cell lines that have an ability to form invadopodia. These results indicate that invadopodia are the sites where enrichment and trafficking of lipid rafts occur and that Cav-1 is an essential regulator of MT1-MMP function and invadopodia-mediated breast cancer cell invasion.

The Role of EF-hand Domains and C2 Domain in Regulation of Enzymatic Activity of Phospholipase Cζ

Sperm-specific phospholipase C-ζ (PLCζ) induces Ca2+ oscillations and egg activation when injected into mouse eggs. PLCζ has such a high Ca2+ sensitivity of PLC activity that the enzyme can be active in resting cells at ∼100 nm Ca2+, suitable for a putative sperm factor to be introduced into the egg at fertilization (Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T., and Miyazaki, S. (2004) J. Biol. Chem. 279, 10408–10412). In the present structure-function analysis, deletion of EF1 and EF2 of the N-terminal four EF-hand domains caused marked reduction of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-hydrolyzing activity in vitro and loss of Ca2+ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant. However, deletion of EF1 and EF2 or mutation of EF1 or EF2 at the x and z positions of the putative Ca2+-binding loop little affected the Ca2+ sensitivity of the PLC activity, whereas deletion of EF1 to EF3 caused 12-fold elevation of the EC50 of Ca2+ concentration. Thus, EF1 and EF2 are important for the PLCζ activity, and EF3 is responsible for its high Ca2+ sensitivity. Deletion of four EF-hand domains or the C-terminal C2 domain caused complete loss of PLC activity, indicating that both regions are prerequisites for PLCζ activity. Screening of interactions between the C2 domain and phosphoinositides revealed that C2 has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P but not to PI(4,5)P2 or acidic phospholipids. PI(3)P and PI(5)P reduced PLCζ activity in vitro, suggesting that the interaction could play a role for negative regulation of PLCζ.

Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation

Inhibition of p110α or of the downstream PI3K signaling pathway components PDK1 and Akt, as well as phosphoinositide sequestration, blocks invadopodia formation in breast cancer cells.

Proteolytic activation of protein kinase C delta and epsilon by caspase-3 in U937 cells during chemotherapeutic agent-induced apoptosis.

Protein kinase C (PKC) family members play pivotal roles in cellular signal transduction and nPKCdelta and theta are known to be subjected to restrictive proteolysis during apoptosis. Here we show that nPKCepsilon was specifically cleaved and generates 43-kDa and 36-kDa C-terminal fragments during chemotherapeutic drug-induced apoptosis. The proteolytic cleavage of nPKCdelta and epsilon was completely inhibited by pretreatment with Ac-DEVD-cho, a specific inhibitor of caspase-3 family enzymes. Furthermore, nPKCepsilon in non-treated U937 cell lysates was cleaved by purified recombinant caspase-3 to generate the 43-kDa fragment, identical in size to the fragment observed in vivo. This cleavage was prevented by the addition of Ac-DEVD-cho. These results suggest that caspase-3 specifically cleaves nPKCepsilon. These findings suggest the possibility that nPKC subfamily members are generally involved in the execution of apoptosis but they are regulated diversely depending on the different apoptotic stimuli.

Phosphatidylinositol 4,5-bisphosphate and PIP5-kinase Iα are required for invadopodia formation in human breast cancer cells

Invadopodia are ventral cell protrusions formed in invasive cancer cells. Because invadopodia have extracellular matrix (ECM) degradation activity, they are thought to function in cancer invasion. In this study, we examined the roles of phosphatidylinositol 4,5‐bisphosphate [PI(4,5)P2] and PI(4,5)P2‐producing enzymes in invadopodia formation in MDA‐MB‐231 human breast cancer cells. Immunofluorescence analysis showed that PI(4,5)P2 accumulates at invadopodia on the ventral cell surface. Injection of an anti‐PI(4,5)P2 antibody inhibited invadopodia formation along with gelatin degradation activity. Sequestering of PI(4,5)P2 by overexpression of the phospholipase C (PLC) δ1‐pleckstrin homology (PH) domain, a specific probe for PI(4,5)P2, also blocked invadopodia formation, while a mutated PLCδ1‐PH domain that lacks PI(4,5)P2‐binding activity had no effect. Cellular PI(4,5)P2 production is mainly mediated by type‐I phosphatidylinositol 4‐phosphate 5‐kinase (PIP5KI) family proteins, which include PIP5KIα, Iβ, and Iγ. Real‐time quantitative PCR analysis showed that PIP5KIα is a dominant isoform expressed in MDA‐MB‐231 cells. Knockdown of PIP5KIα by small‐interfering RNA (siRNA) inhibited invadopodia formation and gelatin degradation. Immunofluorescence analysis revealed that endogenous PIP5KIα protein localizes at invadopodia, which is corroborated by the observation that exogenously expressed green fluorescent protein (GFP)‐fused PIP5KIα protein also accumulates at gelatin degradation sites. These results indicate that localized production of PI(4,5)P2 by PIP5KIα is required for invadopodia formation and ECM degradation by human breast cancer cells. (Cancer Sci 2010)

p120 Catenin Recruits Cadherins to γ-Secretase and Inhibits Production of Aβ Peptide

The γ-secretase complex cleaves many transmembrane proteins, including amyloid precursor protein, EphB and ErbB tyrosine kinase receptors, Notch1 receptors, and adhesion factors. Presenilin 1, the catalytic subunit of γ-secretase, associates with the cadherin/catenin cell-cell adhesion/communication system and promotes cadherin processing (Georgakopoulos, A., et al. (1999) Mol. Cell 4, 893–902; Marambaud, P., et al. (2002) EMBO J. 21, 1948–1956), but the mechanism by which γ-secretase and cadherins associate is unclear. Here we report that p120 catenin (p120ctn), a component of the cadherin-catenin complex, recruits γ-secretase to cadherins, thus stimulating their processing while inhibiting production of Aβ peptide and the amyloid precursor protein intracellular domain. This function of p120ctn depends on both p120ctn-cadherin and p120ctn-presenilin 1 binding, indicating that p120ctn is the central factor that bridges γ-secretase and cadherin-catenin complexes. Our data show that p120ctn is a unique positive regulator of the γ-secretase processing of cadherins and a negative regulator of the amyloid precursor protein processing. Furthermore, our data suggest that specific members of the γ-secretase complex may be used to recruit different substrates and that distinct PS1 sequences are required for processing of APP and cadherins.

Phospholipase Cδ3 Regulates RhoA/Rho Kinase Signaling and Neurite Outgrowth

Phospholipase Cδ3 (PLCδ3) is a key enzyme regulating phosphoinositide metabolism; however, its physiological function remains unknown. Because PLCδ3 is highly enriched in the cerebellum and cerebral cortex, we examined the role of PLCδ3 in neuronal migration and outgrowth. PLCδ3 knockdown (KD) inhibits neurite formation of cerebellar granule cells, and application of PLCδ3KD using in utero electroporation in the developing brain results in the retardation of the radial migration of neurons in the cerebral cortex. In addition, PLCδ3KD inhibits axon and dendrite outgrowth in primary cortical neurons. PLCδ3KD also suppresses neurite formation of Neuro2a neuroblastoma cells induced by serum withdrawal or treatment with retinoic acid. This inhibition is released by the reintroduction of wild-type PLCδ3. Interestingly, the H393A mutant lacking phosphatidylinositol 4,5-bisphosphate hydrolyzing activity generates supernumerary protrusions, and a constitutively active mutant promotes extensive neurite outgrowth, indicating that PLC activity is important for normal neurite outgrowth. The introduction of dominant negative RhoA (RhoA-DN) or treatment with Y-27632, a Rho kinase-specific inhibitor, rescues the neurite extension in PLCδ3KD Neuro2a cells. Similar effects were also detected in primary cortical neurons. Furthermore, the RhoA expression level was significantly decreased by serum withdrawal or retinoic acid in control cells, although this decrease was not observed in PLCδ3KD cells. We also found that exogenous expression of PLCδ3 down-regulated RhoA protein, and constitutively active PLCδ3 promotes the RhoA down-regulation more significantly than PLCδ3 upon differentiation. These results indicate that PLCδ3 negatively regulates RhoA expression, inhibits RhoA/Rho kinase signaling, and thereby promotes neurite extension.

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells.

Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)) has anti-cancer activity in several colon cancers. 1α,25(OH)(2)D(3) induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKIIβ) but not PIPKIIα is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLCδ1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLCδ1 PHD inhibited 1α,25(OH)(2)D(3)-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P(2) production mediates E-cadherin expression through PIPKIIβ in a VDR-dependent manner. PIPKIIβ is also involved in the suppression of the cell motility induced by 1α,25(OH)(2)D(3). These results indicate that PIPKIIβ-mediated PI(4,5)P(2) signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.

Phospholipase C-eta2 is highly expressed in the habenula and retina.

Phospholipase C (PLC), a key enzyme involved in phosphoinositide turnover, hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate two second messengers, inositol 1,4,5-triphosphate and diacylglycerol. PLCeta2 (PLCeta2), a neuron-specific isozyme of PLC, is abundantly expressed in the postnatal brain, suggesting the importance of PLCeta2 in the formation and maintenance of the neuronal network in the postnatal brain. However, the detailed expression patterns of PLCeta2 in the brain and other neuronal tissues remain to be clarified. Here, we generated PLCeta2 knockout/LacZ knockin (plch2(lacZ)(/)(lacZ)) mice-the first mice to lack full-length PLCeta2. Although the plch2(lacZ)(/)(lacZ) mice exhibited no obvious abnormalities, the LacZ reporter revealed unexpected and abundant expressions of PLCeta2 in the habenula and retina. We confirmed these PLCeta2 expression patterns by in situ hybridization and immunohistochemical analyses. In the retina, strong PLCeta2 expression was detected in the photoreceptor (rod/cone), outer nuclear layer, outer plexiform layer, and inner nuclear layer, suggesting that PLCeta2 is expressed in rods and cones, and also in horizontal, bipolar, and amacrine cells, but not in ganglion cells. Interestingly PLCeta2 exhibited a dynamic expression pattern during postnatal retinal development, strongly suggesting that this isozyme might be involved in the development and maturation of the retina. Since both the habenula and retina are thought to play important roles in the regulation of circadian rhythms, our results suggest that PLCeta2 may be involved in the function of habenula and retina.

Monoacylglycerol lipase promotes Fcγ receptor-mediated phagocytosis in microglia but does not regulate LPS-induced upregulation of inflammatory cytokines

Monoacylglycerol lipase (MAGL) is important for neuroinflammation. However, the regulatory mechanisms underlying its expression and function remain unknown. Lipopolysaccharide (LPS) treatment post-translationally upregulated MAGL expression, whereas it downregulated MAGL transcription through a Stat6-mediated mechanism in microglia. Neither MAGL knockdown nor JZL-184, a selective MAGL inhibitor, suppressed LPS-induced upregulation of inflammatory cytokines in microglia. Moreover, exogenous expression of MAGL in BV-2 microglial cell line, which lacks endogenous MAGL, did not promote the induction of inflammatory cytokines by LPS treatment. Interestingly, MAGL knockdown reduced Fcγ receptor-mediated phagocytosis in primary microglia, and introduction of MAGL into the BV-2 cells increased Fcγ receptor-mediated phagocytosis. Collectively, these results suggest that MAGL regulates phagocytosis, but not LPS-mediated cytokine induction in microglia.