Yoshimi Homma

Cutting Edge: Essential Role of Phospholipase C-γ2 in B Cell Development and Function

Cross-linking of the B cell Ag receptor (BCR) induces the tyrosine phosphorylation of multiple cellular substrates, including phospholipase C (PLC)-γ2, which is involved in the activation of the phosphatidylinositol pathway. To assess the importance of PLC-γ2 in murine lymphopoiesis, the PLC-γ2 gene was inducibly ablated by using IFN-regulated Cre recombinase. Mice with a neonatally induced loss of PLC-γ2 function displayed reduced numbers of mature conventional B cells and peritoneal B1 cells and defective responses in vitro to BCR stimulation and in vivo to immunization with thymus-independent type II Ags. In contrast, T cell development and TCR-mediated proliferation were normal. Taken together, PLC-γ2 is a critical component of BCR signaling pathways and is required to promote B cell development.

Essential role of Mannose-binding lectin-associated serine protease-1 in activation of the complement factor D

The complement system is an essential component of innate immunity, participating in the pathogenesis of inflammatory diseases and in host defense. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme; MASP-2 is responsible for the lectin pathway activation. The function of other serine proteases (MASP-1 and MASP-3) is still obscure. In this study, we generated a MASP-1– and MASP-3–deficient mouse model (Masp1/3−/−) and found that no activation of the alternative pathway was observed in Masp1/3−/− serum. Mass spectrometric analysis revealed that circulating complement factor D (Df) in Masp1/3−/− mice is a zymogen (pro-Df) with the activation peptide QPRGR at its N terminus. These results suggested that Masp1/3−/− mice failed to convert pro-Df to its active form, whereas it was generally accepted that the activation peptide of pro-Df is removed during its secretion and factor D constitutively exists in an active form in the circulation. Furthermore, recombinant MASP-1 converted pro-Df to the active form in vitro, although the activation mechanism of pro-Df by MASP-1 is still unclear. Thus, it is clear that MASP-1 is an essential protease of both the lectin and alternative complement pathways.

Morphological Changes and Detachment of Adherent Cells Induced by p122, a GTPase-activating Protein for Rho

We recently cloned a novel signaling molecule, p122, that shows a GTPase-activating activity specific for Rho and the ability to enhance the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C δ1 in vitro. Here we analyzed the in vivo function of p122. Microinjection of the GTPase-activating domain of p122 suppressed the formation of stress fibers and focal adhesions induced by lysophosphatidic acid, suggesting a GTPase-activating activity for Rho as in in vitro. Transfection of p122 also induced the disassembly of stress fibers and the morphological rounding of various adherent cells. Analyses using deletion and point mutants demonstrated that the GTPase-activating domain of p122 is responsible for the morphological changes and detachment and that arginine residues at positions 668 and 710 and a lysine residue at position 706 in the GTPase-activating domain are essential. Using Fluo-3-based Ca2+ microscopy, we found that p122 evoked a rapid elevation of intracellular Ca2+ levels, suggesting that p122 stimulates the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C δ1. These results demonstrate that p122 synergistically functions as a GTPase-activating protein specific for Rho and an activator of phospholipase C δ1 in vivo and induces morphological changes and detachment through cytoskeletal reorganization.

Tissue- and cell type-specific expression of mRNAs for four types of inositol phospholipid-specific phospholipase C

The mRNA levels for four types of inositol phospholipid-specific phospholipase C (PLC) in various tissues and cell cultures have been studied by Northern analysis using cDNA probes for PLC isozyme I, II, and III [Sue, P.-G., Ryu, S.H., Moon, K.H., Sue, H.W., and Rhee, S.G. (1988) Proc. Natl. Acad. Sci. USA 85, 5419-5423 and Cell 54, 161-169], and the recently identified isozyme IV. All four types are ubiquitously expressed in rat tissues, but the levels of the mRNAs vary among tissues and cell lines. PLC-I mRNA levels are extremely high in brain and rat C6 glioma cells with lower levels in other tissues tested. PLC-II and -III have a more widespread distribution, with relatively high levels in brain, lung, spleen, thymus, and testis in the case of PLC-II, and in skeletal muscle, spleen, and testis for PLC-III. PLC-II and -III mRNAs were also detected in all cell lines examined except human promyelocytic HL60 cells. PLC-IV mRNA levels are extraordinarily high in spleen and HL60 cells. These results indicate that rat C6 glioma cells, together with most rat tissues, contain all four PLC isozymes. Other cultured cell types examined also contain two or three PLC isozymes except for HL60 cells, which contain only PLC-IV. The concomitant expression of PLC isozymes in cultured cells suggests a diverse function for PLC isozymes in single cells.

An Invasion-Independent Pathway of Blood-Borne Metastasis: A New Murine Mammary Tumor Model

It is generally believed that active invasion by cancer cells is essential to the metastatic process. In this report, we describe a murine mammary tumor (MCH66) model of metastasis that does not require invasion into the vascular wall of both the primary tumor and the target organ, in this case, the lung. The process involves intravasation of tumor nests surrounded by sinusoidal blood vessels, followed by intravascular tumor growth in the lung, without penetration of the vascular wall during the process. Comparative studies using a nonmetastatic MCH66 clone (MCH66C8) and another highly invasive metastatic cell line (MCH416) suggested that high angiogenic activity and sinusoidal remodeling of tumor blood vessels were prerequisites for MCH66 metastasis. Differential cDNA analysis identified several genes that were overexpressed by MCH66, including genes for the angiogenesis factor pleiotrophin, and extracellular matrix-associated molecules that may modulate the microenvironment toward neovascularization. Our analyses suggest that tumor angiogenesis plays a role in the induction of invasion-independent metastasis. This model should prove useful in screening and development of new therapeutic agents for cancer metastasis.

Characterization of the interaction between RhoA and the amino-terminal region of PKN

The yeast two‐hybrid system and in vitro binding assay were carried out to characterize the interaction between PKN and a small GTP‐binding protein, RhoA. It was revealed that the region corresponding to the amino acid residues 33–111 in the amino‐terminal region of PKN was sufficient to confer the ability to associate with RhoA. Each synthetic peptide fragment corresponding to the amino acid residues 74–93 and 94–113 of PKN inhibited the interaction between PKN and RhoA in the in vitro binding assay, suggesting that this region is important in the association with RhoA. The endogenous and the GAP‐stimulated GTPase activity of RhoA was inhibited by the interaction with PKN, suggesting the presence of a regulatory mechanism that sustains the GTP‐bound active form of RhoA.

Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein

Mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis coli and also sporadic colorectal cancer development. By using antibodies raised against the N-terminal region of APC protein, we have detected the variable masses of endogenous APC proteins in individual cell lines established from human colorectal carcinomas caused by nonsense mutations of the gene. Phosphorylation of immunoprecipitates of full-length and truncated APC were observed in in vitro kinase reaction, indicating association of APC with protein kinase activity. The kinase activity complexed with APC was sensitive to heparin and used GTP as phosphoryl donor, suggesting an involvement of casein kinase 2 (CK2). Both CK2α- and β-subunits were found to associate with APC in immunoprecipitates as well as in pull-down assays, with preferential interaction of APC with tetrameric CK2 holoenzyme. In synchronized cell populations, the association of APC with CK2 was cell cycle dependent, with the highest association in G2/M. Unexpectedly, APC immunoprecipitates containing full-length APC protein inhibited CK2 in vitro, whereas immunoprecipitates of truncated APC had little effect. This was confirmed by using recombinant APC, and the inhibitory region was localized to the C terminus of APC between residues 2086 and 2394. Overexpression of this fragment in SW480 cells suppressed cell proliferation rates as well as tumorigenesis. These results demonstrate a previously uncharacterized functional interaction between the tumor suppressor protein APC and CK2 and suggest that growth-inhibitory effects of APC may be regulated by inhibition of CK2.

Suppression of corneal neovascularization by culture supernatant of human amniotic cells.

Purpose. To examine the applicability of culture supernatant of human amniotic cells on basic fibroblast growth factor (bFGF)–induced corneal neovascularization. Methods. Human amniotic epithelial and mesenchymal cells (AC) were obtained from human amniotic membranes by digesting with collagenase and maintained in serum-containing medium. The AC preparations predominantly contained cytokeratin-positive cells (91.2 ± 3.1%, n = 4). The culture supernatant was prepared by cultivating AC in serum-free medium for 24 hours. Neovascularization was obtained by a micropocket assay with Hydron pellets containing bFGF. Migration assay was carried out by a double-chamber method using human umbilical vein endothelial cells (HUVEC). Cell growth assay was done by MTT assay using HUVEC. Results. Basic fibroblast growth factor–induced corneal neovascularization was significantly reduced by administration of AC culture supernatant. When either control or AC culture supernatant was administered three times per day for 10 days, the area with neovascularization was 13.2 ± 3.2 mm 2 and 4.0 ± 1.4 mm 2 for the control and AC culture supernatant–treated eyes, respectively (n = 7, p = 0.021). AC culture supernatant strongly inhibited bFGF-induced migration and cell growth of HUVEC. Conclusions. Amniotic cell culture supernatant contains potent inhibitors of neovascularization. This effect is explained in part by inhibition of migration and cell growth of vascular endothelial cells. AC culture supernatant may be applicable for treatment of corneal diseases with neovascularization.

Involvement of a Novel Zinc Finger Protein, MIZF, in Transcriptional Repression by Interacting with a Methyl-CpG-binding Protein, MBD2

MBD2, a methyl-CpG-binding protein, is a component of the MeCP1 histone deacetylase (HDAC) complex and plays a critical role in DNA methylation-mediated transcriptional repression. To understand the molecular basis of the methylation-associated repression, we attempted to identify MBD2-interacting proteins by a yeast two-hybrid system. Using MBD2 as bait, we isolated a novel zinc finger protein, referred to as MIZF. A direct interaction between MBD2 and MIZF was confirmed by in vitro binding assays and immunoprecipitation experiments. Four of seven zinc fingers present in the C-terminal region of MIZF are required for binding with MBD2. The MIZF mRNA is expressed in all human tissues and cell lines examined. The subcellular localization of MIZF is distinct from that of MBD2, although both proteins co-localize in some areas of the nuclei; MIZF localizes diffusely in the nucleoplasmic region, whereas MBD2 preferentially localizes in major satellites. A reporter assay demonstrated that MIZF significantly abrogates transcriptional activities. This repression is attenuated by an HDAC inhibitor, trichostatin A, and is completely dependent on the interaction with MBD2. These results suggest that MIZF is abundantly present in cells and functions as a negative regulator of transcription by binding to MBD2 and recruiting HDAC-containing complexes.

Involvement of EphB1 receptor/EphrinB2 ligand in neuropathic pain.

Study Design. We investigated involvement of EphB/ephrinB system in neuropathic pain. Objective. Using immunoblotting, immunohistochemistry, and RNA interference techniques, we examined the expression levels of EphB receptors and ephrinB ligands in neuropathic pain. We also explored the effect of ephrinB siRNA for neuropathic pain. Summary of Background Data. It has been reported that EphB2 regulates the development of synaptic plasticity in the hippocampus by interacting with N-methyl-D-aspartate (NMDA) receptors. In acute pain models, it has been clear that EphB1/ephrinB2 interactions via the NMDA receptor modulates synaptic efficacy in spinal cord. Methods. Adult female Sprague-Dawley rats were used in this study. A crush injury model was prepared by crushing the left L5 spinal nerve distal to dorsal root ganglions (DRG) under deep anesthesia. The sham operation was subjected as control. Expression of ephrinB2 and EphB1 were examined by immunoblotting and immunohistochemical analyses with anti-EphB and anti-ephrinB antibodies. To assess involvement of ephrinB in neuropathic pain, we examined the effect of small interference RNA (siRNA) on mechanical allodynia. Results. Among EphB and ephrinB isoforms tested, ephrinB2 and EphB1 were predominant in DRG and spinal cord. Results showed that the expression of ephrinB2 was enhanced in neurons in DRG and spinal cord by the injury in a time-dependent manner. EphB1 was expressed in neurons of spinal cord. Administration of ephrinB2 siRNA reduced the expression of ephrinB2 and mechanical allodynia. Conclusion. Expression of ephrinB2 is enhanced by nerve injury in neurons in DRG and spinal cord, while its receptor EphB1 is expressed in spinal cord. These results suggest that induction of ephrinB2 might activate EphB1/ephrinB2 signaling pathway to regulate synaptic plasticity and reorganization, and that ephrinB2 siRNA could be a potential therapeutic agent for neuropathic pain.