The dachs gene was first identified almost a century ago based on its requirements for appendage growth, but has been relatively little studied. Here, we describe the phenotypes of strong dachs mutations, report the cloning of the dachs gene, characterize the localization of Dachs protein, and investigate the relationship between Dachs and the Fat pathway. Mutation of dachs reduces, but does not abolish, the growth of legs and wings. dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells. dachs mutations suppress the effects of fat mutations on gene expression, cell affinity and growth in imaginal discs. Dachs protein localization is influenced by Fat, Four-jointed and Dachsous, consistent with its genetic placement downstream of fat. However, dachs mutations have only mild tissue polarity phenotypes, and only partially suppress the tissue polarity defects of fat mutants. Our results implicate Dachs as a crucial downstream component of a Fat signaling pathway that influences growth, affinity and gene expression during development.
The role of cytosolic phospholipase A 2 (cPLA 2 ) in the regulation of ceramide formation was examined in a cell line (L929) responsive to the cytotoxic action of tumor necrosis factor ␣ (TNF␣). In L929 cells, the addition of TNF␣ resulted in the release of arachidonate, which was followed by a prolonged accumulation of ceramide occurring over 5-12 h and reaching 250% over base line. The formation of ceramide was accompanied by the hydrolysis of sphingomyelin and the activation of three distinct sphingomyelinases (neutral Mg 2؉ -dependent, neutral Mg 2؉ -independent, and acidic enzymes). The variant cell line C12, which lacks cPLA 2 , is resistant to the cytotoxic action of TNF␣. TNF␣ was able to activate nuclear factor B in both the wild-type L929 cells and the C12 cells. However, TNF␣ was unable to cause the release of arachidonate or the accumulation of ceramide in C12 cells. C 6 -ceramide overcame the resistance to TNF␣ and caused cell death in C12 cells to a level similar to that in L929 cells. The introduction of the cPLA 2 gene into C12 cells resulted in partial restoration of TNF␣-induced arachidonate release, ceramide accumulation, and cytotoxicity. This study suggests that cPLA 2 is a necessary component in the pathways leading to ceramide accumulation and cell death.The sphingomyelin (SM) 1 cycle, first described by Okazaki et al.(1), has gained recognition over the past few years as a key mechanism for regulating anti-mitogenic signals. Activation of this cycle through the regulation of a signal-induced sphingomyelinase (SMase) results in generation of the lipid second messenger ceramide. Ceramide then modulates a number of biological fates, including growth inhibition (1-3), differentiation (2), apoptosis (4 -6), and cell cycle arrest (7). Although recent studies have begun to catalogue inducers such as TNF␣, interleukin-1, nerve growth factor, and Fas that are capable of signaling through the SM cycle (see Refs. 5, 6, and 8 for reviews), the mechanisms by which these inducers stimulate SMase activity remain poorly understood.TNF␣, through interaction with either a 55-or 75-kDa TNF receptor (9, 10), impacts upon a myriad of intracellular signaling cascades, including protein phosphorylation cascades, transcription factors, and lipid messengers (11). Two classes of lipid mediators have been implicated in TNF␣ signaling, glycerophospholipid metabolites and sphingolipid metabolites (11,12), and recent evidence suggests that these two classes of lipids may interact (13). In HL-60 cells, a linear correlation was established among TNF␣ stimulation, AA generation, and SM cycle activation: TNF␣-stimulated AA liberation preceded ceramide generation, and AA reproduced the effects of TNF␣ on the SM cycle (13). Although these studies suggested that AA and/or its metabolites may be involved in activation of SMase, the physiologic role of the PLA 2 /AA pathway in regulating SMase activity has not been determined.In this study, we examined the role of PLA 2 in SMase activation in the L929 murine fibroblast cell l...
Both glutathione (GSH) depletion and arachidonic acid (AA) generation have been shown to regulate sphingomyelin (SM) hydrolysis and are known components in tumor necrosis factor K K (TNFK K)-induced cell death. In addition, both have hypothesized direct roles in activation of N-sphingomyelinase (SMase); however, it is not known whether these are independent pathways of N-SMase regulation or linked components of a single ordered pathway. This study was aimed at differentiating these possibilities using L929 cells. Depletion of GSH with Lbuthionin-(S,R)-sulfoximine (BSO) induced 50% hydrolysis of SM at 12 h. In addition, TNF induced a depletion of GSH, and exogenous addition of GSH blocked TNF-induced SM hydrolysis as well as TNF-induced cell death. Together, these results establish GSH upstream of SM hydrolysis and ceramide generation in L929 cells. We next analyzed the L929 variant, C12, which lacks both cytosolic phospholipase A 2 (cPLA 2 ) mRNA and protein, in order to determine the relationship of cPLA 2 and GSH. TNF did not induce a significant drop in GSH levels in the C12 line. On the other hand, AA alone was capable of inducing a 60% depletion of GSH in C12 cells, suggesting that these cells remain responsive to AA distal to the site of cPLA 2 . Furthermore, depleting GSH with BSO failed to effect AA release, but caused a drop in SM levels, showing that the defect in these cells was upstream of the GSH drop and SMase activation. When cPLA 2 was restored to the C12 line by expression of the cDNA, the resulting CPL4 cells regained sensitivity to TNF. Treatment of the CPL4 cells with TNF resulted in GSH levels dropping to levels near those of the wildtype L929 cells. These results demonstrate that GSH depletion following TNF treatment in L929 cells is dependent on intact cPLA 2 activity, and suggest a pathway in which activation of cPLA 2 is required for the oxidation and reduction of GSH levels followed by activation of SMases. ß 2000 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
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