Toyohiro Tada

Hyperosmolar Mannitol Stimulates Expression of Aquaporins 4 and 9 through a p38 Mitogen-activated Protein Kinase-dependent Pathway in Rat Astrocytes*

The membrane pore proteins, aquaporins (AQPs), facilitate the osmotically driven passage of water and, in some instances, small solutes. Under hyperosmotic conditions, the expression of some AQPs changes, and some studies have shown that the expression of AQP1 and AQP5 is regulated by MAPKs. However, the mechanisms regulating the expression of AQP4 and AQP9 induced by hyperosmotic stress are poorly understood. In this study, we observed that hyperosmotic stress induced by mannitol increased the expression of AQP4 and AQP9 in cultured rat astrocytes, and intraperitoneal infusion of mannitol increased AQP4 and AQP9 in the rat brain cortex. In addition, a p38 MAPK inhibitor, but not ERK and JNK inhibitors, suppressed their expression in cultured astrocytes. AQPs play important roles in maintaining brain homeostasis. The expression of AQP4 and AQP9 in astrocytes changes after brain ischemia or traumatic injury, and some studies have shown that p38 MAPK in astrocytes is activated under similar conditions. Since mannitol is commonly used to reduce brain edema, understanding the regulation of AQPs and p38 MAPK in astrocytes under hyperosmotic conditions induced with mannitol may lead to a control of water movements and a new treatment for brain edema.

Immunohistochemical localization of Zn-alpha 2-glycoprotein in normal human tissues.

The Zn-alpha 2-glycoprotein (Zn-alpha 2-GP) is present at a high concentration in the seminal plasma and at significant levels in other human body fluids. Its precise localization, however, has remained unclear, as well as its physiological and pathological significance. The present study reports the immunohistochemical localization of this protein in normal adult human tissues. Localization of the reactive product to anti-human plasma Zn-alpha 2-GP antibody was demonstrated in the following cells: luminal and basal cells of the prostate gland, luminal epithelial cells of the acini and of some ducts of the mammary glands, luminal cells of the secretory portion of the eccrine and apocrine sweat glands, serous cells of the salivary, tracheal, and bronchial glands, acinar cells of the esophageal glands, exocrine acinar cells of the pancreas, hepatocytes of the liver, and epithelial cells of the proximal and distal tubules in the kidney. The present results suggest that Zn-alpha 2-GP exerts some unknown but fairly widespread exocrine function and may be produced in the various epithelial cells tested. Hepatocytes are also suggested to be a source of the protein in the blood plasma.

Rapid translocation of cytosolic Ca2+/calmodulin-dependent protein kinase II into postsynaptic density after decapitation.

Abstract: The postsynaptic density (PSD) fraction prepared from rat forebrains frozen with liquid nitrogen immediately after dissection (within 30 s after decapitation) contained major postsynaptic density protein (mPSDp), α subunit of Ca2+/calmodulin‐dependent protein kinase II (CaMKII) at a level of merely 2.7% of the total protein. The content of the protein in the fraction was increased to ∼10% by placing the forebrains on ice for a few minutes. Accumulation, but to a lesser extent, of the protein after placement was also observed in the particulate, synaptosome, and synaptic plasma membrane fractions with its concomitant decrease in the cytosolic fraction. The distribution change may be translocation of the protein, because the amounts of the losses of the protein in the cytosolic fraction were balanced by the gains in the particulate fractions. By translocation, CaMKII became Triton X‐100 insoluble and partially inactivated. The amount of CaMKII transferred from the cytosol to particulate fractions at 0°C was about the same as that contained in the conventional PSD fraction. Furthermore, the thickness of the PSD was increased by the treatment of the forebrains at 37°C, by which the content of CaMKIIα in the PSD fraction was increased to twofold. These results suggest that most of the CaMKII α subunit associated with the PSD fraction (mPSDp) is translocated from cytosol after decapitation. We also showed similar translocation of CaMKIIβ/β′.

Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenation

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens [Cell 39 (1984) 49], that increase plasma membrane water permeability in secretory and absorptive cells. In the central nervous system (CNS), we detected the transcripts of AQP3, 5 and 8 in addition to the previously reported transcripts of AQP4 and 9 in astrocytes, of AQP3, 5 and 8 in neurons, of AQP8 in oligodendrocytes, and none of them in microglia using RNase protection assay and the reverse transcription-polymerase chain reaction (RT-PCR). Hypoxia evoked a marked decrease in the expression levels of AQP4, 5 and 9, but not of AQP3 and 8 mRNAs, and in astrocytes in vitro subsequent reoxygenation elicited the restoration of the expression of AQP4 and 9 to their basal levels. Interestingly, AQP5 showed a transient up-regulation (about 3-fold) and subsequent down-regulation of its expression within 20 h of reoxygenation after hypoxia. The changes in the profiles of AQP expression during hypoxia and reoxygenation were also observed by Western blot analysis. These results suggest that AQP5 may be one of the candidates for inducing the intracranial edema in the CNS after ischemia injury.

Usefulness of D2-40 immunohistochemistry for differentiation between kaposiform hemangioendothelioma and tufted angioma.

Background:  Recent investigations have demonstrated the utility of the monoclonal antibody D2‐40 as a marker for lymphatic endothelium. D2‐40 can be used on formalin‐fixed and paraffin‐embedded materials. Our objective was to elucidate, using D2‐40 immunohistochemistry, the differences among capillary hemangiomas, and especially between kaposiform hemangioendothelioma (KHE) and tufted angioma (TA). We studied four cases of KHE, nine cases of TA, and 31 cases of other vascular tumors. Antibodies against CD31, CD34, factor VIII‐related antigen, and GLUT1 were also applied.

Interleukin‐1β induces the expression of aquaporin‐4 through a nuclear factor‐κB pathway in rat astrocytes

Interleukin (IL)‐1β is known to play a role in the formation of brain edema after various types of injury. Aquaporin (AQP)4 is also reported to be involved in the progression of brain edema. We tested the hypothesis that AQP4 is induced in response to IL‐1β. We found that expression of AQP4 mRNA and protein was significantly up‐regulated by IL‐1β in cultured rat astrocytes, and that intracerebroventricular administration of IL‐1β increased the expression of AQP4 protein in rat brain. The effects of IL‐1β on induction of AQP4 were concentration and time dependent. The effects of IL‐1β on AQP4 were mediated through IL‐1β receptors because they were abolished by co‐incubation with IL‐1 receptor antagonist. It appeared that IL‐1β increased the level of AQP4 mRNA without involvement of de novo protein synthesis because cycloheximide, a protein synthesis inhibitor, did not inhibit the effects of IL‐1β. Inhibition of the nuclear factor‐κB (NF‐κB) pathway blocked the induction of AQP4 by IL‐1β in a concentration‐dependent manner. These findings show that IL‐1β induces expression of AQP4 through a NF‐κB pathway without involvement of de novo protein synthesis in rat astrocytes.

Induction of Notch signaling by tumor necrosis factor in rheumatoid synovial fibroblasts.

Rheumatoid arthritis (RA) is characterized by progressive inflammation associated with abberrant proliferation of synoviocytes. In order to explore the characteristics of rheumatoid synovial fibroblasts (RSF), we performed the comparative gene expression profile analysis between RSF and normal synovial fibroblasts (NSF) upon tumor necrosis factor (TNF) stimulation. As an initial screening for the genes preferentially induced by TNF in RSF compared with NSF, we have adopted a cDNA array containing well-defined sets of genes responsible for cell growth, cell fate determination, and cellular invasiveness. Differentially expressed genes of interest were confirmed using real-time RT–PCR. We found that TNF induced the expression of Notch-1, Notch-4, and Jagged-2 in RSF. The expression of these proteins was detected in the RA synovial tissues. The nucleus of RA synoviocytes showed strong staining with anti-Notch-1 and Notch-4 antibody. TNF induced the nuclear translocation of Notch intracellular domain in RSF, indicating the elicitation of the Notch signaling. Notch-1, Notch-4, and Jagged-2 proteins were also detected in the developing synovium of mouse embryo. Thus, RSF may have re-acquired the primordial phenotype, accounting for the hyperproliferation and aggressive invasiveness, exhibiting tumor-like phenotype.

Experimental implication of celiac ganglionotropic invasion of pancreatic-cancer cells bearing c-ret proto-oncogene with reference to glial-cell-line-derived neurotrophic factor (GDNF)

Perineural invasion is a prominent clinical feature of pancreatic cancer which causes difficulty in curative resection. In the present study, the human pancreatic cancer cell lines, PaCa‐2, AsPC‐1, SW1990 and Capan‐2, were all found to express abundant c‐ret proto‐oncogene mRNA and RET protein, a member of the receptor‐tyrosine‐kinase superfamily, identified as being a receptor for glial‐cell‐line‐derived neurotrophic factor (GDNF). In an invasion assay, the migration of pancreatic cancer cells was markedly induced by co‐cultivation with human glioma cells, T98G or A172, capable of producing and secreting GDNF. Anti‐GDNF antibody in conditioned media of glioma cells suppressed much of the migratory activity. Checkerboard analysis of the migration showed both chemotactic and chemokinetic activity of GDNF. There was no detectable expression of another GDNF receptor component, a glycosyl‐phosphatidylinositol‐linked receptor (GFRα‐1), in pancreatic‐cancer cell lines, suggesting that the neural invasion of pancreatic‐cancer cells spreads along a concentration gradient of GDNF produced from peripheral ganglions through direct interaction of GDNF with its receptor, the c‐ret proto‐oncogene product. Immunochemical localization of GDNF in human celiac ganglionic tissue supported this contention. Int. J. Cancer 81:67–73, 1999.

Alpha-fetoprotein producing gastric cancer lacks transcription factor ATBF1.

Alpha-fetoprotein (AFP) producing gastric cancer (AFP–GC) is very malignant and highly metastatic compared with common gastric cancer. However, the causal relationship between AFP production and the high malignancy of AFP-GC is unclear. We investigated AFP gene regulation in AFP-GC by an active transcription factor, HNF1 (hapatocyte nuclear factor 1) and a repressive transcription factor, ATBF1 (AT motif binding factor 1). RNase protection assays revealed that the production of AFP in gastric cancer cells did not directly associate with HNF1 expression. An inverse relation between the expressions of ATBF1 and AFP was clearly observed in gastric cancer cells. CAT assays showed the direct inhibition of AFP gene expression by ATBF1. Methylation analysis of the AFP promoter region in gastric cancer cells suggested that methylation itself could not explain the silencing of the AFP gene. Immunohistochemistry of resected clinical samples revealed that AFP producing cells lacked ATBF1 immunoreactivity. Our data suggests that the absence of ATBF1 is responsible for AFP gene expression in gastric cancer, and the absence of ATBF1 is a distinct characteristic of AFP-GC and might be important for its highly malignant nature.

Absence of Procarboxypeptidase R Induces Complement-Mediated Lethal Inflammation in Lipopolysaccharide-Primed Mice

Carboxypeptidase R (CPR) is a heat-labile enzyme found in serum in addition to stable carboxypeptidase N. CPR cleaves the C-terminal basic amino acids, arginine and lysine, from inflammatory peptides such as complement C3a and C5a, bradykinin, and enkephalin. This enzyme is generated from procarboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor, following cleavage by proteolytic enzymes such as thrombin, plasmin, and trypsin. We generated proCPR-deficient mice by knocking out exons 4 and 5 of the proCPR gene, which are regarded as essential for CPR function. At LPS challenge, there was virtually no difference in lethality among proCPR+/+, proCPR+/−, and proCPR−/− mice. However, challenge with cobra venom factor, which can activate and deplete almost all complement in vivo, induced a lethal effect on proCPR−/− mice following LPS sensitization which up-regulates C5a receptor expression. In contrast, proCPR+/+ and proCPR+/− mice were able to tolerate the cobra venom factor challenge with the limited dose (30 U). Although carboxypeptidase N plays a role in inactivation of inflammatory peptides in vivo, CPR may also be important in the regulation of hyperinflammation.