Katsuhiko Nakashima

Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes.

Peptidylarginine deiminase (PAD) deiminates arginine residues in proteins to citrulline residues Ca2+ dependently. There are four types of PADs, I, II, III, and V, in humans. We studied the subcellular distribution of PAD V in HL-60 granulocytes and peripheral blood granulocytes. Expression of green fluorescent protein-tagged PADs in HeLa cells revealed that PAD V is localized in the nucleus, whereas PAD I, II, and III are localized in the cytoplasm. PAD V deletion mutants indicated that the sequence residues 45–74 have a nuclear localization signal (NLS). A sequence feature of this NLS is a three-lysine residue cluster preceded by a proline residue and is not found in the three other PADs. Substitution of the lysine cluster by an alanine cluster abrogated the nuclear import activity. These results suggested that the NLS is a classical monopartite NLS. HL-60 granulocytes, neutrophils, and eosinophils stained with antibody specific for PAD V exhibited distinct positive signals in the nucleus. Subcellular fractionation of HL-60 granulocytes also showed the nuclear localization of the enzyme. When neutrophils were stimulated with calcium ionophore A23187, protein deimination occurred in the nucleus. The major deiminated proteins were identified as histones H2A, H3, and H4. The implication of PAD V in histone modifications is discussed.

Structural basis for Ca(2+)-induced activation of human PAD4

Peptidylarginine deiminase 4 (PAD4) is a Ca2+-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. Its gene is a susceptibility locus for rheumatoid arthritis. Here we present the crystal structure of Ca2+-free wild-type PAD4, which shows that the polypeptide chain adopts an elongated fold in which the N-terminal domain forms two immunoglobulin-like subdomains, and the C-terminal domain forms an α/β propeller structure. Five Ca2+-binding sites, none of which adopt an EF-hand motif, were identified in the structure of a Ca2+-bound inactive mutant with and without bound substrate. These structural data indicate that Ca2+ binding induces conformational changes that generate the active site cleft. Our findings identify a novel mechanism for enzyme activation by Ca2+ ions, and are important for understanding the mechanism of protein citrullination and for developing PAD-inhibiting drugs for the treatment of rheumatoid arthritis.

Molecular Characterization of Peptidylarginine Deiminase in HL-60 Cells Induced by Retinoic Acid and 1α,25-Dihydroxyvitamin D3

Three types of peptidylarginine deiminase (PAD), which converts a protein arginine residue to a citrulline residue, are widely distributed in animal tissues. Little is known about PAD of hemopoietic cells. We found that PAD activity in human myeloid leukemia HL-60 cells was induced with the granulocyte-inducing agents retinoic acid and dimethyl sulfoxide and with the monocyte-inducing agent 1α,25-dihydroxyvitamin D3. We cloned and characterized a PAD cDNA from retinoic acid-induced cells. The cDNA was 2,238 base pairs long and encoded a 663-amino acid polypeptide. The HL-60 PAD had 50–55% amino acid sequence identities with the three known enzymes and 73% identity with the recently cloned keratinocyte PAD. The recombinant enzyme differs in kinetic properties from the known enzymes. Immunoblotting and Northern blotting with an antiserum against the enzyme and the cDNA, respectively, showed that a protein of approximately 67 kDa increased concomitantly with increase of mRNA of approximately 2.6 kilobases during granulocyte differentiation. During monocyte differentiation the same mRNA and protein increased as in granulocyte differentiation. Neither the enzyme activity nor the protein was found in macrophage-induced cells. These results suggested that expression of the PAD gene is tightly linked to myeloid differentiation.

Immunocytochemical localization of peptidylarginine deiminase in human eosinophils and neutrophils

Peptidylarginine deiminase, registered as PAD V in the DDBJ/GenBank/EMBL data banks, is expressed in HL‐60 cells differentiated into granulocytes or monocytes. We analyzed PAD activities in density‐fractionated human peripheral blood cell fractions. PAD activity with similar substrate specificity to that of PAD V was found in the eosinophil and neutrophil fractions, which showed single bands comigrating with authentic PAD V on immunoblotting with an anti‐PAD V antibody. Both the biochemical and immunoblotting analyses showed marked enrichment of PAD V in the eosinophil fraction. Its immunoreactivity appeared to localize in eosinophilic granules at high density and in myeloperoxidase‐negative cytoplasmic granules of neutrophils at low density, as determined by confocal laser‐scanning microscopy. Possible roles of PAD V in myeloid differentiation and granulocyte function are discussed. In addition, we present evidence for the presence of PAD(s) that are antigenically different from PAD V in monocytes and lymphocytes.

SND1, a Component of RNA-Induced Silencing Complex, Is Up-regulated in Human Colon Cancers and Implicated in Early Stage Colon Carcinogenesis

Colon cancers have been shown to develop after accumulation of multiple genetic and epigenetic alterations with changes in global gene expression profiles, contributing to the establishment of widely diverse phenotypes. Transcriptional and posttranscriptional regulation of gene expression by small RNA species, such as the small interfering RNA and microRNA and the RNA-induced silencing complex (RISC), is currently drawing major interest with regard to cancer development. SND1, also called Tudor-SN and p100 and recently reported to be a component of RISC, is among the list of highly expressed genes in human colon cancers. In the present study, we showed remarkable up-regulation of SND1 mRNA in human colon cancer tissues, even in early-stage lesions, and also in colon cancer cell lines. When mouse Snd1 was stably overexpressed in IEC6 rat intestinal epithelial cells, contact inhibition was lost and cell growth was promoted, even after the cells became confluent. Intriguingly, IEC6 cells with high levels of Snd1 also showed an altered distribution of E-cadherin from the cell membrane to the cytoplasm, suggesting loss of cellular polarity. Furthermore, the adenomatous polyposis coli (Apc) protein was coincidentally down-regulated, with no significant changes in the Apc mRNA level. Immunohistochemical analysis using chemically induced colonic lesions developed in rats revealed overexpression of Snd1 not only in colon cancers but also in aberrant crypt foci, putative precancerous lesions of the colon. Up-regulation of SND1 may thus occur at a very early stage in colon carcinogenesis and contribute to the posttranscriptional regulation of key players in colon cancer development, including APC and beta-catenin.

Oncogenic Ras/ERK Signaling Activates CDCP1 to Promote Tumor Invasion and Metastasis

Involvement of Ras in cancer initiation is known, but recent evidence indicates a role in cancer progression, including metastasis and invasion; however, the mechanism is still unknown. In this study, it was determined that human lung cancer cells with Ras mutations, among other popular mutations, showed significantly higher expression of CUB domain–containing protein 1 (CDCP1) than those without. Furthermore, activated Ras clearly induced CDCP1, whereas CDCP1 knockdown or inhibition of CDCP1 phosphorylation by Src-directed therapy abrogated anoikis resistance, migration, and invasion induced by activated-Ras. Activation of MMP2 and secretion of MMP9, in a model of Ras-induced invasion, was found to be regulated through induction of phosphorylated CDCP1. Thus, CDCP1 is required for the functional link between Ras and Src signaling during the multistage development of human malignant tumors, highlighting CDCP1 as a potent target for treatment in the broad spectrum of human cancers associated with these oncogenes. Implications: CDCP1 protein induced by oncogenic Ras/Erk signaling is essential for Ras-mediated metastatic potential of cancer cells. Mol Cancer Res; 12(10); 1449–59. ©2014 AACR.

Dynamic Expression of Peptidylarginine Deiminase 2 in Human Monocytic Leukaemia THP-1 Cells During Macrophage Differentiation

Peptidylarginine deiminases (PADs) consist of five enzymes which are widely distributed in human and rodent tissues. The two types of enzymes are found in human peripheral blood cells; PAD4 mainly in granulocytes and monocytes and PAD2 in lymphocytes and macrophages. Little is known about the regulation of PAD expression in macrophages. Here, we report that PAD2 is expressed in human monocytic leukaemia THP-1 cells during differentiation into macrophages by 12-O-tetradecanoylphorbol-13-acetate. During this differentiation, the levels of PAD2 mRNA and protein increased concomitantly, indicating the transcriptional regulation of PAD2 gene expression in the cells. The treatment of THP-1-derived macrophages with calcium ionophore A23187 generated vimentin deimination and resulted in the disruption of vimentin filament organization. We discuss the possible role of vimentin deimination in cell physiology.

Augmentation of invadopodia formation in temozolomide-resistant or adopted glioma is regulated by c-Jun terminal kinase-paxillin axis.

Temozolomide (TMZ) is one of the few effective anticancer agents against gliomas. However, acquisition of TMZ resistance or adaptation by gliomas is currently a crucial problem, especially increased invasiveness which is critical for the determination of clinical prognosis. This study investigated the molecular regulatory mechanisms of TMZ resistance in gliomas involved in invasiveness, particularly invadopodia formation, a molecular complex formed at the invasive front to cause extracellular matrix degradation during cellular local invasion. The TMZ-resistant clone of the U343 MG human glioma cell line (U343-R cells) was established. U343-R cells demonstrated higher invadopodia formation compared with U343 cells without TMZ resistance (U343-Con cells). Immunoblot analysis of DNA damage-related mitogen-activated protein kinase signals found increased phosphorylation of c-Jun terminal kinase (JNK) and higher activation of its downstream signaling in U343-R cells compared with U343-Con cells. Treatment of U343-R cells with specific inhibitors of JNK or siRNA targeting JNK suppressed up-regulation of invadopodia formation. In addition, paxillin, one of the known JNK effectors which is phosphorylated and affects cell migration, was phosphorylated at serine 178 in JNK activity-dependent manner. Expression of paxillin with mutation of the serine 178 phosphorylation site in U343-R cells blocked invadopodia formation. The present findings suggest that increased formation of invadopodia in U343-R cells is mediated by hyperactivation of JNK-paxillin signaling, and both JNK and paxillin might become targets of novel therapies against TMZ-resistant gliomas.

Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft

Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism.

Novel small molecule inhibiting CDCP1‐PKCδ pathway reduces tumor metastasis and proliferation

CUB domain‐containing protein‐1 (CDCP1) is a trans‐membrane protein predominantly expressed in various cancer cells and involved in tumor progression. CDCP1 is phosphorylated at tyrosine residues in the intracellular domain by Src family kinases and recruits PKCδ to the plasma membrane through tyrosine phosphorylation‐dependent association with the C2 domain of PKCδ, which in turn induces a survival signal in an anchorage‐independent condition. In this study, we used our cell‐free screening system to identify a small compound, glycoconjugated palladium complex (Pd‐Oqn), which significantly inhibited the interaction between the C2 domain of PKCδ and phosphorylated CDCP1. Immunoprecipitation assays demonstrated that Pd‐Oqn hindered the intercellular interaction of phosphorylated CDCP1 with PKCδ and also suppressed the phosphorylation of PKCδ but not that of ERK or AKT. In addition, Pd‐Oqn inhibited the colony formation of gastric adenocarcinoma 44As3 cells in soft agar as well as their invasion. In mouse models, Pd‐Oqn markedly reduced the peritoneal dissemination of gastric adenocarcinoma cells and the tumor growth of pancreatic cancer orthotopic xenografts. These results suggest that the novel compound Pd‐Oqn reduces tumor metastasis and growth by inhibiting the association between CDCP1 and PKCδ, thus potentially representing a promising candidate among therapeutic reagents targeting protein–protein interaction.