Diverse cellular localizations of secretory phospholipase A2 enzymes in several human tissues

The secretory phospholipase A2 (sPLA2) family in mammals contains more than 10 enzymes. In this study, we examined by immunohistochemistry the localization of six sPLA2s (IIA, IID, IIE, IIF, V and X) in human heart, kidney, liver and stomach. In normal hearts, sPLA2-IIA was detected in coronary vascular smooth muscle cells (VSMC) and sPLA2-V in cardiomyocytes beneath the endocardium. In infarcted hearts, expression of these two enzymes was markedly increased in damaged cardiomyocytes, and expression of sPLA2-IID and-IIE, which was undetectable in normal hearts, was elevated in damaged cardiomyocytes and VSMC, respectively. In infarcted kidneys, sPLA2-IIA and-V were markedly induced in the uriniferous tubular epithelium. In livers affected by viral hepatitis, sPLA2-IIA and-V were expressed in hepatocytes with fatty degeneration. In the gastric glands exhibiting intestinal metaplasia, sPLA2-IIA was localized in the glandular base, sPLA2-IID and-V in the glandular body epithelium, sPLA2-IIE and-IIF in goblet cells in the foveolar epithelium, and sPLA2-X in both glandular body epithelial cells and foveolar epithelial goblet cells. In the gastric submucosal tissues, sPLA2-IIA and-IIE were located in VSMC and sPLA2-V was in the interstitial fibroblasts. In addition, sPLA2-IIA,-IIE,-IIF and-X were highly expressed in gastric signet ring cell carcinoma. Thus, individual sPLA2s exhibit unique cellular localizations in each tissue, suggesting their distinct roles in pathophysiology.     

S100 proteins modulate protein phosphatase 5 function: a link between CA2+ signal transduction and protein dephosphorylation

PP5 is a unique member of serine/threonine phosphatases comprising a regulatory tetratricopeptide repeat (TPR) domain and functions in signaling pathways that control many cellular responses. We reported previously that Ca(2+)/S100 proteins directly associate with several TPR-containing proteins and lead to dissociate the interactions of TPR proteins with their client proteins. Here, we identified protein phosphatase 5 (PP5) as a novel target of S100 proteins. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, and S100B proteins specifically interact with PP5-TPR and inhibited the PP5-Hsp90 interaction. In addition, the S100 proteins activate PP5 by using a synthetic phosphopeptide and a physiological protein substrate, Tau. Overexpression of S100A1 in COS-7 cells induced dephosphorylation of Tau. However, S100A1 and permanently active S100P inhibited the apoptosis signal-regulating kinase 1 (ASK1) and PP5 interaction, resulting the inhibition of dephosphorylation of phospho-ASK1 by PP5. The association of the S100 proteins with PP5 provides a Ca(2+)-dependent regulatory mechanism for the phosphorylation status of intracellular proteins through the regulation of PP5 enzymatic activity or PP5-client protein interaction.     

A Subclone of HuH-7 with Enhanced Intracellular Hepatitis C Virus Production and Evasion of Virus Related-Cell Cycle Arrest

Hepatitis C virus (HCV) cell culture system with JFH-1 strain and HuH-7 cells enabled us to produce infectious HCV particles in vitro, and such system is useful to explore the anti-HCV compounds and to develop the vaccine against HCV. In the present study, we describe the derivation of a cell line that permits improved production of HCV particles. Specifically, we characterized several subclones that were isolated from the original HuH-7 cell line by limiting dilution. These HuH-7 subclones displayed a notable range of HCV production levels following transfection by full-genome JFH-1 RNA. Among these subclones, HuH-7T1 produced HCV more efficiently than other subclones and Huh-7.5.1 that is known to be highly permissive for HCV replication. Upon transfection with full-genome RNA, HCV production was increased ten-fold in HuH-7T1 compared to Huh-7.5.1. This increase in viral production correlated with increased efficiency of intracellular infectious virus production. Furthermore, HCV replication did not induce cell cycle arrest in HuH-7T1, whereas it did in Huh-7.5.1. Consequently, the use of HuH-7T1 as host cells could provide increased population of HCV-positive cells and elevated viral titer. In conclusion, we isolated a HuH-7 subclone, HuH-7T1, that supports efficient HCV production. High efficiency of intracellular infectious virus production and evasion of cell cycle arrest were important for this phenotype. We expect that the use of this cell line will facilitate analysis of the underlying mechanisms for HCV particle assembly and the cell cycle arrest caused by HCV.     

Pirfenidone inhibits the expression of HSP47 in TGF-beta1-stimulated human lung fibroblasts

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and patients with idiopathic pulmonary fibrosis (IPF). Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen and plays an important role in the pathogenesis of IPF. The present study evaluated the in vitro effects of pirfenidone on expression of HSP47 and collagen type I in cultured normal human lung fibroblasts (NHLF). Expression levels of HSP47 and collagen type I in NHLF stimulated by transforming growth factor (TGF)-beta1 were evaluated genetically, immunologically and immunocytochemically. Treatment with TGF-beta1 stimulated both mRNA and protein expressions of both HSP47 and collagen type I in NHLF, and pirfenidone significantly inhibited this TGF-beta1-enhanced expression in a dose-dependent manner. We concluded that the anti-fibrotic effect of pirfenidone may be mediated not only through direct inhibition of collagen type I expression but also at least partly through inhibition of HSP47 expression in lung fibroblasts, with a resultant reduction of collagen synthesis in lung fibrosis.     

Human group III secreted phospholipase A2 promotes neuronal outgrowth and survival

Human sPLA2-III [group III secreted PLA2 (phospholipase A2)] is an atypical sPLA2 isoenzyme that consists of a central group III sPLA2 domain flanked by unique N- and C-terminal domains. In the present study, we found that sPLA2-III is expressed in neuronal cells, such as peripheral neuronal fibres, spinal DRG (dorsal root ganglia) neurons and cerebellar Purkinje cells. Adenoviral expression of sPLA2-III in PC12 cells (pheochromocytoma cells) or DRG explants facilitated neurite outgrowth, whereas expression of a catalytically inactive sPLA2-III mutant or use of sPLA2-III-directed siRNA (small interfering RNA) reduced NGF (nerve growth factor)-induced neuritogenesis. sPLA2-III also suppressed neuronal death induced by NGF deprivation. Lipid MS revealed that sPLA2-III overexpression increased the cellular level of lysophosphatidylcholine, a PLA2 reaction product with neuritogenic and neurotropic activities, whereas siRNA knockdown reduced the level of lysophosphatidylcholine. These observations suggest the potential contribution of sPLA2-III to neuronal differentiation and its function under certain conditions.