Type I platelet-activating factor acetylhydrolase catalytic subunits over-expression induces pleiomorphic nuclei and centrosome amplification.

LIS1, a causative gene product for type I lissencephaly, binds to and regulates the dynein motor and the centrosome. LIS1 also forms a complex with the catalytic subunits alpha1 and alpha2 of type I platelet-activating factor acetylhydrolase [PAF-AH (I)]. However, the cellular function of the catalytic subunits remains unknown. In this study, we showed that over-expression of the catalytic subunits, especially alpha2, in cultured cells induced dramatic phenotypical changes including nuclear shape change, centrosomal amplification and microtubule disorganization. We examined if these effects were due to the catalytic activity and/or binding of alpha2 to LIS1. Substitution of a single amino acid Glu39 of murine alpha1 and alpha2 by Asp (alpha2-E39D) did not affect catalytic activity but completely abolished LIS1 binding. Over-expression of either alpha2-E39D or the catalytically inactive alpha2-S48C revealed that alpha2-E39D, but not alpha2-S48C, lost its ability to induce above-mentioned phenotypic changes. Biochemical analyses showed that LIS1 present in the precipitate fraction of murine brain homogenates could be translocated to the soluble fraction by alpha2, but not by alpha2-E39D. These results suggest that over-expression of the PAF-AH (I) catalytic subunits induces centrosomal amplification and microtubule disorganization by disturbing intracellular localization of LIS1.

Biochemical and molecular characterization of a novel choline-specific glycerophosphodiester phosphodiesterase belonging to the nucleotide pyrophosphatase/phosphodiesterase family.

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that release nucleoside 5'-monophosphate from a variety of nucleotides and nucleotide derivatives. The mammalian NPP family comprises seven members, but only three of these (NPP1-3) have been studied in some detail. Previously we showed that lysophospholipase D, which hydrolyzes lysophosphatidylcholine (LPC) to produce lysophosphatidic acid, is identical to NPP2. More recently an uncharacterized novel NPP member (NPP7) was shown to have alkaline sphingomyelinase activity. These findings raised the possibility that other members of the NPP family act on phospholipids. Here we show that the sixth member of the NPP family, NPP6, is a choline-specific glycerophosphodiester phosphodiesterase. The sequence of NPP6 encodes a transmembrane protein containing an NPP domain with significant homology to NPP4, NPP5, and NPP7/alkaline sphingomyelinase. When expressed in HeLa cells, NPP6 was detected in both the cells and the cell culture medium as judged by Western blotting and by enzymatic activity. Recombinant NPP6 efficiently hydrolyzed the classical substrate for phospholipase C, p-nitrophenyl phosphorylcholine, but not the classical nucleotide phosphodiesterase substrate, p-nitrophenyl thymidine 5'-monophosphate. In addition, NPP6 hydrolyzed LPC to form monoacylglycerol and phosphorylcholine but not lysophosphatidic acid, showing it has a lysophospholipase C activity. NPP6 showed a preference for LPC with short (12:0 and 14:0) or polyunsaturated (18:2 and 20:4) fatty acids. It also hydrolyzed glycerophosphorylcholine and sphingosylphosphorylcholine efficiently. In mice, NPP6 mRNA was predominantly detected in kidney with a lesser expression in brain and heart, and in human it was detected in kidney and brain. The present results suggest that NPP6 has a specific role through the hydrolysis of polyunsaturated LPC, glycerophosphorylcholine, or sphingosylphosphorylcholine in these organs.

Role of mast cells in the development of renal fibrosis: use of mast cell-deficient rats.

BACKGROUND: Recent clinical studies have shown that the number of interstitial mast cells increases in various types of renal disease and correlates well with the magnitude of interstitial fibrosis. The present study was conducted to assess the role of mast cells in renal fibrosis by examining an experimental glomerular disease. METHODS: A rat model of chronic glomerular disease, puromycin aminonucleoside-nephrosis, was induced in mast cell-deficient (Ws/Ws) and normal (+/+) rats. RESULTS: The area of interstitial fibrosis was widely distributed at 6 weeks in both groups of rats; however, unexpectedly, the area of interstitial fibrosis was greater in Ws/Ws rats than in +/+ littermates. Biochemical analysis of the hydroxyproline content confirmed the more severe fibrosis in the Ws/Ws rats. The number of mast cells increased in both Ws/Ws and +/+ rats, concomitant with the development of interstitial fibrosis, but was confirmed to be lower in Ws/Ws than in +/+ rats. There were no differences in the numbers of interstitial macrophages and T lymphocytes between the two groups. Reverse transcription-polymerase chain reaction analysis of cytokine expression revealed that the level of mRNA for transforming growth factor-beta (TGF-beta), a potent profibrotic cytokine, was higher in Ws/Ws rats. In addition, heparin, one of the major components of mast cells, inhibited the expression of TGF-beta mRNA in rat fibroblasts in culture. CONCLUSION: These results suggest that mast cells do not play a major role in the pathogenesis of interstitial fibrosis in puromycin aminonucleoside nephrosis. Rather, they might be protective or ameliorative in this model through the inhibition of TGF-beta production by heparin, and possibly in other models and also in humans.

Induction of cytokines by toxins that have an identical RNA N-glycosidase activity: Shiga toxin, ricin, and modeccin.

Shiga toxin (Stx) has an A1-B5 subunit structure, and the A subunit is an RNA N-glycosidase that inhibits cellular protein synthesis. We previously reported that in Caco-2 cells Stx induced cytokines and that the RNA N-glycosidase activity was essential for the cytokine induction. It is known that the binding of the Stx-B subunit to its receptor glycolipid, Gb3, mediates an A subunit-independent signal in some types of cells, but the involvement of this signal in the cytokine induction is unclear. In this study, we investigated whether RNA N-glycosidase itself induces cytokines. IL-8 production was enhanced by Stx, ricin, and modeccin, three toxins that inhibit protein synthesis through an identical RNA N-glycosidase activity, but not by two other types of protein synthesis inhibitors, diphtheria toxin and cycloheximide. The RNA N-glycosidase-type toxins showed a similar induction pattern of cytokine mRNAs. Brefeldin A, a Golgi apparatus inhibitor, completely suppressed the cytokine induction by the toxins. Analysis by using inhibitors of toxin binding and also Stx-B subunit showed that the cytokine-inducing activity was independent of Gb3-mediated signaling. These results indicate that RNA N-glycosidase itself induces the cytokine production and that intracellular transport of toxins through the Golgi apparatus is essential for the activity.

Altered expression of NDST-1 messenger RNA in puromycin aminonucleoside nephrosis.

Sulfated portions of glycosaminoglycan (GAG) side chains in heparan sulfate proteoglycan (HSPG) are thought to play an important role in charge-dependent selectivity of glomerular filtration against plasma proteins. Heparan sulfate N-acetylglucosamine N-deacetylase/adenosine 3'-phosphate 5'-phosphosulfate: unsubstituted glucosamine N-sulfotransferase (NDST) is the key enzyme regulating sulfation of GAG chains. In this study we investigated transcriptional expression of NDST-1, 1 of 4 isozymes of NDST, in glomeruli of rats with puromycin aminonucleoside (PAN) nephrosis. Nephrosis was induced in rats with a single intraperitoneal injection of 150 mg/kg PAN. On days 10 and 35, expression of NDST-1 messenger RNA (mRNA) in glomeruli was analyzed with the use of Northern-blot analysis. Immunohistochemical studies were also performed with the use of monoclonal antibodies that react specifically with the N-sulfated portion of the GAG chain of HSPG and agrin, a major core protein of HSPG in glomerular basement membrane (GBM). In addition, we studied the expression of NDST-1 mRNA in cultured glomerular epithelial cells (GECs) and glomerular mesangial cells in the presence of PAN. On day 10, when significant proteinuria developed, the ratios of glomerular expression of NDST-1 mRNA against glyceraldehyde-phosphate dehydrogenase mRNA in PAN-treated rats were decreased to 48% +/- 6% of those in controls (P<.05). Immunohistochemical studies revealed that staining for N-sulfated GAG chains of HSPG on GBM was markedly reduced on day 10 in PAN-treated rats but that staining for agrin was unchanged. In contrast, on day 35, when PAN-treated rats recovered from proteinuria, we noted no differences in glomerular expression of NDST-1 mRNA and staining intensity for N-sulfated GAG chains on GBM between PAN-treated rats and controls. Incubation of GECs for 24 hours in the presence of 50 ng/mL PAN resulted in the reduction of the expression of NDST-1 mRNA (67% +/- 12% of those in controls, P<.05). In summary, we found alteration of the expression of NDST-1 mRNA, accompanying a loss of N-sulfated GAG chains of HSPG on GBM without changes in the core protein agrin, in the course of PAN nephrosis. These data suggest an important role for this enzyme in heparan sulfate assembly in GBM and GEC and in the pathogenesis of proteinuria in PAN nephrosis.

Enhanced expression of C chemokine lymphotactin in IgA nephropathy.

Leukocyte accumulation in the kidney is observed in patients with IgA nephropathy. Chemokines are a large family of cytokines chemotactic for leukocytes and have been shown to be upregulated in renal diseases. We previously reported that the gene expression of lymphotactin, a sole member of C chemokine subfamily, is enhanced in an animal model of crescentic glomerulonephritis, but its expression in human renal diseases is totally unknown. In the present study, we investigated the expression of mRNAs of lymphotactin and some other chemokines in IgA nephropathy. The expression of mRNAs for three chemokines, lymphotactin, MCP-1, and MIP-1beta, in renal cortex was increased and the levels of lymphotactin and MCP-1 mRNAs were statistically higher in patients with glomerular crescents than in those without crescents. These levels also correlated with tubulointerstitial changes and urinary protein excretion. Glomerular levels of mRNAs for lymphotactin and MCP-1, but not MIP-1beta, were higher in IgA nephropathy than controls. By immunohistochemical analysis, lymphotactin was detected in tryptase-positive cells (putative mast cells) in the interstitial space. These results suggest that lymphotactin, as well as MCP-1, may contribute to leukocyte infiltration and disease progression in IgA nephropathy.

Fractalkine expression and the recruitment of CX3CR1+ cells in the prolonged mesangial proliferative glomerulonephritis.

BACKGROUND: We established the reversible and the prolonged models of mesangial proliferative glomerulonephritis (GN) with anti-Thy 1 antibody 1-22-3. However, the essential factors leading to the prolonged glomerular alterations have not been identified. METHODS: The expressions of several chemokines and cytokines were compared in the reversible and the prolonged models. Expression of fractalkine and the number of the fractalkine receptor CX3CR1-positive cells in the glomeruli in the prolonged model were significantly higher than those in the reversible model. Then, the localization of fractalkine and the characteristics of CX3CR1+ cells were analyzed in glomeruli. To elucidate the significance of the fractalkine expression, we analyzed the expression in the model treated with angiotensin II receptor antagonist, candesartan. RESULTS: Immunostaining of fractalkine was detected on endothelial cells on the fifth day, and fractalkine staining also was detected in the mesangial area on day 14. Major parts of the CX3CR1+ cells in the glomeruli were macrophages, especially ED3+ cells. Candesartan treatment ameliorated the glomerular morphological findings at six weeks after disease induction. Although the treatment did not ameliorate the morphological finding at two weeks, decreased expression of fractalkine and CX3CR1+ were already detected at two weeks in rats treated with candesartan. CONCLUSIONS: Fractalkine expression and the recruitment of CX3CR1+ cells in glomeruli might play an important role in the development of the prolonged disease. These expressions could be predictors of the prolonged disease of the mesangial proliferative glomerulonephritis.