Norie Araki

beta 2-Microglobulin modified with advanced glycation end products is a major component of hemodialysis-associated amyloidosis.

beta 2-Microglobulin (beta 2M) is a major constituent of amyloid fibrils in hemodialysis-associated amyloidosis, a complication of long-term hemodialysis patients. Amyloid fibril proteins were isolated from connective tissues forming carpal tunnels in hemodialysis patients with carpal tunnel syndrome. Two-dimensional polyacrylamide gel electrophoresis and Western blotting demonstrated that most of the beta 2M forming amyloid fibrils exhibited a more acidic pI value than normal beta 2M. This acidic beta 2M was also found in a small fraction of beta 2M in sera and urine from these patients, whereas heterogeneity was not observed in healthy individuals. We purified acidic and normal beta 2M from the urine of long-term hemodialysis patients and compared their physicochemical and immunochemical properties. Acidic beta 2M, but not normal beta 2M, was brown in color and fluoresced, both of which are characteristics of advanced glycation end products (AGEs) of the Maillard reaction. Immunochemical studies showed that acidic beta 2M reacted with anti-AGE antibody and also with an antibody against an Amadori product, an early product of the Maillard reaction, but normal beta 2M did not react with either antibody. Incubating normal beta 2M with glucose in vitro resulted in a shift to a more acidic pI, generation of fluorescence, and immunoreactivity to the anti-AGE antibody. The beta 2M forming amyloid fibrils also reacted with anti-AGE antibody. These data provided evidence that AGE-modified beta 2M is a dominant constituent of the amyloid deposits in hemodialysis-associated amyloidosis.

Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway

CD44 is a widely distributed cell surface adhesion molecule and is implicated in diverse biological processes. However, the nature of intracellular signaling triggered by CD44 remains to be elucidated. Here, we show that CD44 undergoes sequential proteolytic cleavage in the ectodomain and intracellular domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. Consequently, CD44ICD acts as a signal transduction molecule, where it translocates to the nucleus and activates transcription mediated through the 12-O-tetradecanoylphorbol 13-acetate–responsive element, which is found in numerous genes involved in diverse cellular processes. Expression of an uncleavable CD44 mutant as well as metalloprotease inhibitor treatment blocks CD44-mediated transcriptional activation. In search of the underlying mechanism, we have found that CD44ICD potentiates transactivation mediated by the transcriptional coactivator CBP/p300. Furthermore, we show that cells expressing CD44ICD produce high levels of CD44 messenger RNA, suggesting that the CD44 gene is one of the potential targets for transcriptional activation by CD44ICD. These observations establish a novel CD44 signaling pathway and shed new light on the functional link between proteolytic processing of an adhesion molecule at the cell surface and transcriptional activation in the nucleus.

AM-3K, an Anti-macrophage Antibody, Recognizes CD163, a Molecule Associated with an Anti-inflammatory Macrophage Phenotype

CD163 is a member of the scavenger receptor cysteine-rich superfamily restricted to the monocyte/macrophage lineage and is thought to be a useful marker for anti-inflammatory or alternatively activated macrophages. In this study we used mass spectrometric analysis to determine that the antigen recognized by the antibody AM-3K, which we previously generated as a tissue macrophage-specific monoclonal antibody, was CD163. An anti-inflammatory subtype of macrophages stimulated by dexamethasone or interleukin-10 showed strong reactivity for AM-3K and increased expression of CD163 mRNA. Immunohistochemical staining of routinely processed pathological specimens revealed that AM-3K recognized a specialized subpopulation of macrophages. In granulomatous diseases such as tuberculosis, sarcoidosis, or foreign body reactions, tissue macrophages around granulomas, but not component cells of the granulomas such as epithelioid cells and multinucleated giant cells, showed positive staining for AM-3K. In atherosclerotic lesions, scattered macrophages in diffuse intimal lesions were strongly positive for AM-3K, whereas foamy macrophages in atheromatous plaques demonstrated only weak staining. We therefore suggest that, in routine pathological specimens, AM-3K is a useful marker for anti-inflammatory macrophages because these cells can be distinguished from inflammatory or classically activated macrophages. Because AM-3K cross-reacts with macrophage subpopulations in different animal species including rats, guinea pigs, rabbits, cats, dogs, goats, pigs, bovine species, horses, monkeys, and cetaceans, it will have wide application for detection of CD163 in various animals.

Proteomic analysis of human brain identifies α-enolase as a novel autoantigen in Hashimoto’s encephalopathy

Hashimotos encephalopathy (HE) is a rare autoimmune disease associated with Hashimotos thyroiditis (HT). To identify the HE‐related autoantigens, we developed a human brain proteome map using two‐dimensional electrophoresis and applied it to the immuno‐screening of brain proteins that react with autoantibodies in HE patients. After sequential MALDI‐TOF‐MASS analysis, immuno‐positive spots of 48 kDa (pI 7.3–7.8) detected from HE patient sera were identified as a novel autoimmuno‐antigen, α‐enolase, harboring several modifications. Specific high reactivities against human α‐enolase were significant in HE patients with excellent corticosteroid sensitivity, whereas the patients with fair or poor sensitivity to the corticosteroid treatment showed less reactivities than cut‐off level. Although a few HT patients showed faint reactions to α‐enolase, 95% of HT patients, patients with other neurological disorders, and healthy subjects tested were all negative. These results suggest that the detection of anti‐α‐enolase antibody is useful for defining HE‐related pathology, and this proteomic strategy is a powerful method for identifying autoantigens of various central nervous system diseases with unknown autoimmune etiologies.

Calcium influx triggers the sequential proteolysis of extracellular and cytoplasmic domains of E-cadherin, leading to loss of β-catenin from cell – cell contacts

Cadherins are major cell – cell adhesion molecules in both tumor and normal tissues. Although serum levels of soluble E-cadherin have been shown to be higher in the cancer patients than in healthy volunteers, the detail mechanism regulating release of soluble E-cadherin remains to be elucidated. Here we show that the ectodomain of E-cadherin is proteolytically cleaved from some cancer cells by a membrane-bound metalloprotease to yield soluble form, and the residual membrane-tethered cleavage product is subsequently degraded by intracellular proteolytic pathway. Futhermore, we show that extracellular calcium influx, that is induced by mechanical scraping of cells or ionomycin treatment, enhances the metalloprotease-mediated E-cadherin cleavage and the subsequent degradation of the cytoplasmic domain. Immunocytochemical analysis demonstrates that the sequential proteolysis of E-cadherin triggered by the calcium influx results in translocation of β-catenin from the cell – cell contacts to cytoplasm. Our data suggest that calcium influx-induced proteolysis of E-cadherin not only disrupts the cell – cell adhesion but also activates β-catenin-mediated intracellular signaling pathway, potentially leading to alterations in motility and proliferation activity of cells.

Interaction of NE-dlg/SAP102, a Neuronal and Endocrine Tissue-specific Membrane-associated Guanylate Kinase Protein, with Calmodulin and PSD-95/SAP90 A POSSIBLE REGULATORY ROLE IN MOLECULAR CLUSTERING AT SYNAPTIC SITES

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends ofN-methyl-d-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic α-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a K d value of 44 nm. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.

Advanced Glycation End Products and Their Recognition by Macrophage and Macrophage-Derived Cells

Modification of proteins by long-term incubation with glucose leads to the formation of advanced glycation end products (AGEs). AGE proteins are taken up by macrophages via the AGE receptor, which is similar to the macrophage scavenger receptor (MSR). In the present study, we compared the ligand specificity of the AGE receptor with that of MSR by three different experiments. The endocytic uptake of 125I-acetyl-LDL by RAW cells was effectively inhibited by unlabeled AGE-bovine serum albumin (BSA), whereas the inhibitory effect of acetyl-LDL on 125I-AGE-BSA was partial. Polyanions showing an effective inhibition for endocytic uptake of AGE-BSA were not always inhibitory for endocytic degradation of acetyl-LDL. These data, together with those obtained by three-dimensional fluorescence-activated cell sorter analysis, indicate that AGE proteins are recognized by more than two receptors, of which MSR is at least one. Finally, we examined whether MSR could mediate the endocytic uptake of AGE proteins by Chinese hamster ovary cells overexpressing bovine type II MSR (CHO-SRII cells). 125I-AGE-BSA underwent endocytic degradation by CHO-SRII cells, and this was effectively inhibited by unlabeled acetyl-LDL. These results clearly show that MSR mediates the endocytic uptake of AGE proteins, suggesting a new role of MSR in biological recognition of AGE in vivo.

Neurofibromatosis Type 1 (NF1) Tumor Suppressor, Neurofibromin, Regulates the Neuronal Differentiation of PC12 Cells via Its Associating Protein, CRMP-2

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, a negative regulator of Ras. Neurofibromin is implicated in the neuronal abnormality of NF1 patients; however, the precise cellular function of neurofibromin has yet to be clarified. Using proteomic strategies, we identified a set of neurofibromin-associating cellular proteins, including axon regulator CRMP-2 (Collapsin response mediator protein-2). CRMP-2 directly bound to the C-terminal domain of neurofibromin, and this association was regulated by the manner of CRMP-2 phosphorylation. In nerve growth factor-stimulated PC12 cells, neurofibromin and CRMP-2 co-localized particularly on the distal tips and branches of extended neurites. Suppression of neurofibromin using NF1 small interfering RNA significantly inhibited this neurite outgrowth and up-regulated a series of CRMP-2 phosphorylations by kinases identified as CDK5, GSK-3b, and Rho kinase. Overexpression of the NF1-RAS-GAP-related domain rescued these NF1 small interfering RNA-induced events. Our results suggest that neurofibromin regulates neuronal differentiation by performing one or more complementary roles. First, neurofibromin directly regulates CRMP-2 phosphorylation accessibility through the complex formation. Also, neurofibromin appears to indirectly regulate CRMP-2 activity by suppressing CRMP-2-phosphorylating kinase cascades via its Ras-GAP function. Our study demonstrates that the functional association of neurofibromin and CRMP-2 is essential for neuronal cell differentiation and that lack of expression or abnormal regulation of neurofibromin can result in impaired function of neuronal cells, which is likely a factor in NF1-related pathogenesis.

The neurofibromatosis type 1 gene product neurofibromin enhances cell motility by regulating actin filament dynamics via the Rho-ROCK-LIMK2-cofilin pathway

Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients.

Advanced Glycation End Products of the Maillard Reaction in Aortic Pepsin-Insoluble and Pepsin-Soluble Collagen From Diabetic Rats

Recent immunohistological studies using antibodies against advanced glycation end products (AGEs) have demonstrated the presence of AGEs in several tissues. By an enzyme-linked immunosorbent assay using the monoclonal anti-AGE antibody, the present study aimed to determine AGEs in pepsin-insoluble collagen (PIC) as well as in pepsin-soluble collagen (PSC) from the aortas of streptozotocin (STZ)-induced diabetic rats (at 4, 16, and 28 weeks after STZ injection) and those of agematched control rats. Addition of EDTA to the immunoassay buffer has led us to successful determination of AGEs in the aortic PIC samples with following results: 1) in diabetic rats, there was a time-related increase in the AGE contents at 28 weeks (n = 9, 226.4 ± 13.5 ng/mg collagen [mean ± SE]), compared with that at 4 and 16 weeks (n = 6, 79.6 ± 9.5 ng/mg collagen, and n = 8, 149.4 ± 30.9 ng/mg collagen at 4 and 16 weeks, respectively; both P < 0.05, between 4 and 16 weeks and 28 weeks); 2) after 28 weeks of diabetes, the AGE contents in PIC of aortas were significantly higher in diabetic rats than in controls (n = 9, 226.4 ± 13.5 ng/mg collagen vs. n = 8, 129.6 ± 14.9 ng/mg collagen, P < 0.01, diabetic vs. control); and 3) the level of the AGE content was strongly correlated with the PIC/total collagen (TC) ratio (n = 45, r = 0.698, P = 0.0001). By treating the samples of PSC with alkaline solution, the AGE content of PSC was also determined. In the PSC fraction, the AGE levels in the diabetic rats tended to increase with time and to be higher than those of control rats at 28 weeks although these changes were not statistically significant (diabetic: n = 4, 19.4 ± 9.7; n = 6, 22.3 ± 6.2; n = 6, 39.6 ± 10.8; control: n = 4,19.7 ± 9.8; n = 6, 22.9 ± 7.3; n = 7, 30.7 ± 7.2; at 4, 16, and 28 weeks, respectively). Compared with the AGE levels of PSC, those of PIC were about four to seven times and four to five times higher in diabetic and control rats, respectively (PIC versus PSC in diabetic or control rats, all P < 0.001, at 4, 16, and 28 weeks, respectively). These findings provide the first immuno chemical evidence that AGE adducts are present in the materials extracted sequentially by pepsin and collagenase and that these adducts in PIC accumulated as a function of the increase in the aortic PIC/TC ratio.