Shunji Yunoue

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.

PKA phosphorylation and 14-3-3 interaction regulate the function of neurofibromatosis type I tumor suppressor, neurofibromin

Neurofibromin, a neurofibromatosis type I (NF1) tumor suppressor gene product, has a domain acting 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. Therefore, regulation of the function of neurofibromin is heavily involved in cell growth and differentiation. In the present study, we identified a novel cellular neurofibromin‐associating protein, 14‐3‐3, which belongs to a highly conserved family of proteins that regulate intracellular signal transduction events in all eukaryotic cells. The interaction of 14‐3‐3 is mainly directed to the C‐terminal domain (CTD) of neurofibromin, and the cAMP‐dependent protein kinase (PKA)‐dependent phosphorylation clustered on CTD‐Ser (2576, 2578, 2580, 2813) and Thr (2556) is required for the interaction. Interestingly, the increased phosphorylation and association of 14‐3‐3 negatively regulate the function of neurofibromin. These findings indicate that PKA phosphorylation followed by 14‐3‐3 protein interaction may modulate the biochemical and biological functions of neurofibromin.

Phosphorylation of neurofibromin by cAMP-dependent protein kinase is regulated via a cellular association of NG,NG-dimethylarginine dimethylaminohydrolase

The neurofibromatosis type 1 (NF1) tumor suppressor (neurofibromin) is thought to play crucial roles in cellular Ras‐ and cAMP‐dependent kinase (PKA)‐associated signals. In this study, we identified a cellular neurofibromin‐associating protein, N G,N G‐dimethylarginine dimethylaminohydrolase (DDAH) that is known as a cellular NO/NOS regulator. The interaction of DDAH was mainly directed to the C‐terminal domain (CTD) and to the cysteine/serine‐rich domain (CSRD) of neurofibromin, coinciding with the regions containing specific PKA phosphorylation sites. DDAH increased PKA phosphorylation of native neurofibromin in a dose‐dependent manner, especially affecting the phosphorylation of CSRD. These findings suggest that the PKA accessibility of neurofibromin was regulated via DDAH interaction, and this regulation may modulate the cellular function of neurofibromin that is implicated in NF1‐related pathogenesis.

Relationship between cytokeratin staining patterns and clinico-pathological features in somatotropinomae

OBJECTIVE Somatotropinomae are classified as densely and sparsely granulated adenomae, which typically exhibit a perinuclear pattern (PP) and a dot pattern (DP) in cytokeratin (CK) immunostaining respectively. Some exhibit a mixed pattern (MP). We studied the relationship between these somatotropinoma subtypes and their clinico-pathological features. METHODS The study population consisted of 141 Japanese acromegalic patients. We evaluated their clinical presentation and their response to provocation tests with TRH and LHRH and to suppression (octreotide) test. Tumour tissues were subjected to immunostaining for CAM-5.2, MIB-1, CD34, E-cadherin (CDH1) and p53 (TP53). In 43 cases (30 non-DP and 13 DP), we analysed gsp mutations (constitutively activating mutations of the G(s)α protein that is encoded by GNAS gene). RESULTS The 141 adenomae were categorised into three subtypes based on their CK staining patterns; 30 (21.3%) exhibited DP, 83 (58.9%) exhibited PP, and 28 (19.9%) exhibited MP. Compared with the other subtypes, DP adenomae were significantly larger, and their E-cadherin expression and response to TRH, LHRH and octreotide challenge were lower. The postoperative cure rate tended to be lower in DP adenomae. gsp mutations were detected in 25 of 43 cases examined (58.1%); 20 of the 30 non-DP (66.7%) and 5 of the 13 DP tumours (38.5%) were affected by the mutation. CONCLUSION DP somatotropinomae exhibit characteristic features. Compared with the non-DP subtypes, DP adenomae manifested a larger tumour size, a lower incidence of abnormal responses to TRH and LHRH challenge, a poor response to octreotide test and a lower expression of E-cadherin. gsp mutation was not exclusive for non-DP somatotropinomae.

Comparative analysis of brain proteins from p53-deficient mice by two-dimensional electrophoresis

p53 is a tumor suppressor protein that regulates many cellular processes including the cell cycle, DNA repair, and apoptosis. It also serves as a critical regulator of neuronal apoptosis in the central nervous system (CNS). To elucidate the role of p53 in the CNS, brain proteins of p53 knock‐out mice (p53−/−) were analyzed by two‐dimensional gel electrophoresis (2‐DE) and compared with those from p53 wild type (p53+/+) mice. Six types of brain tissue (temporal cortex, cerebellum, hippocampus, striatum, olfactory bulb, and cervical spinal cord) and other control tissues (lung and blood) from 18‐week‐old non‐stress‐induced mice were analyzed. The morphology of brains from p53−/− mice appeared to be normal and identical to that of p53+/+ mice, although lungs showed diffuse tumors that may have been caused by p53 deficiency. Comparative 2‐D gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53−/− specific, whereas 12 of 1008 spots from lung tissue were p53−/− specific. N‐terminal amino acid sequence was determined for p53−/− specific proteins. In all brain tissues from p53−/− mice, a newly identified mouse mitochondrial NADH‐ubiquinone oxidoreductase 24 kDa subunit showed decreased expression, and apolipoprotein A1 acidic forms showed increased expression. In addition, brain‐type creatine kinase B chain and tubulin β‐5 N‐terminal fragment were increased in the p53−/− cerebellum, and a new protein in mouse, hydroxyacylglutathione hydrolase (glyoxalase II) was decreased in the temporal cortex of p53−/− mice. The alterations in protein expression identified in this study may imply a p53‐related brain function. This is the first proteomic analysis on the p53−/− mouse brain, and further information based on this study will provide new insights into the p53 function in the CNS.

Clinico-pathological study of meningiomas with haemorrhagic onset.

Summary¶Background. Haemorrhage from meningiomas is rare and the underlying pathophysiological mechanisms remain to be determined. We sought to identify these mechanisms by studying clinical and histological records of 6 meningioma patients treated at our institution.Patients and methods. We retrospectively studied 6 patients with meningiomas whose acute onset was due to haemorrhage. We evaluated clinical features and imaging studies. The vascularity and proliferative nature of these tumours were examined immunohistochemically and tissue factor (TF) immunoreactivity was assessed. For comparison we evaluated 25 non-haemorrhagic meningiomas.Findings. At onset, the haemorrhages mimicked stroke in all 6 patients. On imaging studies, 3 of the haemorrhages were intra- and extratumoural, the other 3 were extratumoural only. Hyperintensity on T2-weighted MRI was a characteristic of these meningiomas. Histologically, they were of 3 subtypes, meningothelial (n=3), transitional (n=2), and anaplastic (n=1). The MIB-1 labelling index of the 5 WHO Grade I meningiomas was 5.8±2.2. The mean number of CD31-positive blood vessels did not differ in haemorrhagic and non-haemorrhagic meningiomas. The TF-positivity rate of haemorrhagic meningiomas was higher than of non-haemorrhagic meningiomas.Interpretation. The proliferative nature of the meningiomas and TF expression in tumour cells may have contributed to the eventual haemorrhage of the meningiomas in our series.

Identification of CD133+ Cells in Pituitary Adenomas

Stem-like cells in tumors are capable of self-renewal and pluri-differentiation; they are thought to play important roles in tumor initiation and maintenance. Stem-like cells in malignant glioma express CD133. We examined samples from human pituitary adenoma, a generally benign neoplasm, for CD133 expression using routine immunohistochemical and biochemical methods. Our study of 70 pituitary adenomas (clinically nonfunctioning adenomas and growth hormone-, prolactin-, adrenocorticotropic hormone-, and thyroid-stimulating hormone-producing adenomas) showed that 18 (25.7%) expressed CD133. This rate was higher in clinically nonfunctioning (33.3%) than functioning adenomas (12.0%) (p = 0.085). Real-time PCR assay revealed the expression of CD133 mRNA in samples immunohistochemically positive for CD133. Neither the patient age and gender, nor the tumor size or postoperative recurrence rate correlated with CD133 positivity. CD133+ cells ubiquitously coexpressed CD34, nestin, and VEGFR2 (KDL1). S-100 and GFAP were not coexpressed with CD133. Chromogranin A, Pit-1, SF-1, and NeuroD1 were immune-negative, indicating that CD133+ cells did not have the potential to differentiate into functional endocrine cells. Our data suggest that the expression of CD133 in pituitary adenomas is related to immature endothelial progenitor cells that may play a role in the neovascularization of pituitary adenomas. Further studies are needed to elucidate the significance of CD133+ cells with respect to neovascularization and their sustainable growth in pituitary adenomas.

Pituitary metastases: current practice in Japan

OBJECT With advancement of cancer treatment and development of neuroimaging techniques, contemporary clinical pictures of pituitary metastases (PMs) must have changed from past reports. The goal of this paper was to elucidate the clinical features of PMs and current clinical practice related to those lesions. In this retrospective study, questionnaires were sent to 87 physicians who had treated PMs in Japan. RESULTS Between 1995 and 2010, 201 patients with PMs were treated by the participating physicians. The diagnosis of PM was histologically verified in 69 patients (34.3%). In the other 132 patients (65.7%), the PM was diagnosed by their physicians based on neuroimaging findings and clinical courses. The most frequent primary tumor was lung (36.8%), followed by breast (22.9%) and kidney (7.0%) cancer. The average interval between diagnosis of primary cancer and detection of PM was 2.8 ± 3.9 (SD) years. Major symptoms at diagnosis were visual disturbance in 30.3%, diabetes insipidus in 27.4%, fatigue in 25.4%, headache in 20.4%, and double vision in 17.4%. Major neuroimaging features were mass lesion in the pituitary stalk (63.3%), constriction of tumor at the diaphragmatic hiatus (44.7%), hypothalamic mass lesion (17.4%), and hyperintensity in the optic tract (11.4%). Surgical treatment was performed in 26.9% of patients, and 74.6% had radiation therapy; 80.0% of patients who underwent radiotherapy had stereotactic radiotherapy. The median survival time was 12.9 months in total. Contributing factors for good prognosis calculated by Cox proportional hazard analysis were younger age, late metastasis to the pituitary gland, smaller PM size, and radiation therapy. The Kaplan-Meier survival was significantly better in patients with breast cancer and renal cell cancer than in those with lung cancer. CONCLUSIONS At the time of this writing, approximately 60% (120/201) of PMs had been treated by stereotactic radiation therapy in Japan. The median survival time was much longer than that reported in past series. To confirm the changes of clinical features and medical practice, a prospective and population-based survey is mandatory.

Immunoreactivity of Wnt5a, Fzd2, Fzd6, and Ryk in glioblastoma: evaluative methodology for DAB chromogenic immunostaining

The aim of this study was to determine the influence of Wnt5a and its receptors on the survival of glioblastoma patients and to determine reliable evaluation methods for immunohistochemistry. Diagnostic specimens from 41 histopathologically confirmed primary glioblastoma patients whose Gd-enhanced tumors had been totally removed were immunohistochemically stained for Wnt5a, Fzd2, Fzd6, and Ryk. The immunoreactivity was evaluated using the following methods: (A) grayscale optical density after color deconvolution, (B) percentage of stained cells, (C) density of stained cells, (D) staining amount (multiplication product of B and C), and (E) staining rank. The data sets of A to E were statistically evaluated by correlation matrix analysis and regression analysis. The influence of the expression of the markers on survival was analyzed using a proportional hazard model. The results of color deconvolution (A) were well correlated with the results of the staining rank (E). In the semiquantitative results (B, C, and D), the staining amount (D) tended to show a better correlation with results of color deconvolution (A). Among all data sets, color deconvolution (A) demonstrated the most preferable fit in a proportional hazard model, and the expression of Fzd2 and Fzd6 was associated with poor prognosis in glioblastoma patients.

TLR4, IL-6, IL-18, MyD88 and HMGB1 are highly expressed in intracranial inflammatory lesions and the IgG4/IgG ratio correlates with TLR4 and IL-6.

We determined distribution of plasma cells and IgG4/IgG index and factors associated with the index in intracranial inflammatory lesions. Specimens of nine patients were analyzed immunohistochemically using antibodies against CD45, CD68, CD3, CD4, CD8, CD20, CD138, lambda chain, kappa chain, IgG, IgG4, IL‐1α, IL‐6, IL‐18, toll‐like receptor (TLR) 2, TLR4, high‐mobility group box 1 (HMGB1), tumor necrosis factor‐alpha (TNF‐α), myeloid differentiation factor 88 (MyD88), and anaplastic lymphoma kinase (ALK). The relationship between all the factors was assessed using Spearmans rank correlation coefficient (ρ). Negative ALK staining was observed in all the patients. Plasma cells were detected in eight patients with varying degrees. The highest number of neutrophils, but no plasma cells, was observed in a patient with the shortest history of inflammation. IgG4/IgG index was independent of the number of plasma cells. The index was relatively highly correlated with IL‐6 (ρ = 0.7271) and TLR4 expression (ρ = 0.7246). IL‐6 expression was highly correlated with TLR4 expression (ρ = 0.8042). IL‐18 was maximally expressed in all the patients. TLR4 expression was strong, but TRL2 expression was weak. Positive HMGB1 staining was observed in all the patients, predominantly in the nuclei, but also in the cytoplasm in four patients. The cytoplasmic expression strongly correlated with IL‐1α expression (ρ = 0.9583). The cytoplasmic colocalization of HMGB1 and IL‐1α was histologically confirmed in cells with collapsing nuclei by the double‐staining method. The IgG4/IgG indexes varied case by case. IL‐6 and TLR4 expressions may influence IgG4/IgG index. The nuclei of cells with both IL‐1α and HMGB1 expressions in the cytoplasm collapse in the cell death stage. The cooperative high expression of TLR4, IL‐6, IL‐18, MyD88 and HMGB1 suggest their critical roles in the inflammation circuit.