Biomedical subjects
R Kolesnick
Publications and source records attributed to R Kolesnick.
Stress signals for apoptosis: ceramide and c-Jun kinase.
Mammalian systems respond to environmental stress by either adapting or undergoing programmed cell death. While there is general agreement that the caspase family of proteases serve as the effectors of the apoptotic death response, the signaling apparatus involved in the decision to activate the caspase system is less clear. In the past few years, the sphingomyelin and c-Jun Kinase (JNK)/Stress-activated Protein Kinase (SAPK) pathways have been linked to the death response in many cellular systems. These signaling systems are found throughout the animal kingdom, and ceramide signaling is conserved through yeast. Since yeast do not undergo apoptosis, the sphingomyelin pathway appears evolutionarily older than the caspase-mediated death programs. While recent reviews by several groups have broadly surveyed ceramide signaling in apoptosis, this paper examines the role of sphingomyelinases and the JNK/SAPK pathway in coordinate signaling of apoptosis.
BAD enables ceramide to signal apoptosis via Ras and Raf-1.
Prior investigations document that proliferative signaling cascades, under some circumstances, initiate apoptosis, although mechanisms that dictate the final outcome are largely unknown. In COS-7 cells, ceramide signals Raf-1 activation through Ras (Zhang, Y., Yao, B., Delikat, S., Bayoumy, S., Lin, X. H., Basu, S., McGinley, M., Chan-Hui, P. Y., Lichenstein, H., and Kolesnick, R. (1997) Cell 89, 63-72), but not apoptosis. However, expression of small amounts of the pro-apoptotic Bcl-2 family member, BAD, conferred ceramide-induced apoptosis onto COS-7 cells. Ceramide signaled apoptosis in BAD-expressing cells by a pathway involving sequentially kinase suppressor of Ras (KSR)/ceramide-activated protein kinase, Ras, c-Raf-1, and MEK1. Downstream, this pathway linked to BAD dephosphorylation at serine 136 by prolonged inactivation of Akt/PKB. Further, mutation of BAD at serine 136 abrogated ceramide signaling of apoptosis. The present study indicates that when ceramide signals through the Ras/Raf cascade, the availability of a single target, BAD, may dictate an apoptotic outcome.
Caenorhabditis elegans contains two distinct acid sphingomyelinases.
Mounting evidence supports a role for acid sphingomyelinase (ASM) in cellular stress signaling. Only murine and human sphingomyelinases have been defined at the molecular level. These enzymes are the products of a conserved gene and at the amino acid level share 82% identity. In this study, we show that the nematode Caenorhabditis elegans possesses two ASMs, termed ASM-1 and ASM-2 encoded by two distinct genes, but lacks detectable neutral sphingomyelinase activity. The C. elegans ASMs are about 30% identical with each other and with the human and murine enzymes. The conserved regions include a saposin-like domain, proline-rich domain, and a putative signal peptide. In addition, 16 cysteines distributed throughout the molecules, and selected glycosylation sites, are conserved. The expression of these genes in C. elegans is regulated during development. Asm-1 is preferentially expressed in the embryo, whereas asm-2 is predominantly expressed in postembryonic stages. When transfected as Flag-tagged proteins into COS-7 cells, ASM-1 is found almost entirely in a secreted form whereas only 20% of ASM-2 is secreted. Only the secreted forms display enzymatic activity. Furthermore, ASM-2 requires addition of Zn2+ to be fully active, whereas ASM-1 is active in the absence of cation. C. elegans is the first organism to display two ASMs. This finding suggests the existence of an ASM gene family.
12-O-tetradecanoylphorbol-13-acetate-induced apoptosis in LNCaP cells is mediated through ceramide synthase.
Protein kinase C (PKC) activation is often antiapoptotic, although in a few cell types PKC initiates apoptosis by an unknown mechanism. Recent investigations showed that activation of PKC alpha by 12-O-tetradecanoylphorbol 13-acetate (TPA) induced apoptosis in LNCaP prostate cancer cells. The present studies examine the mechanism of this effect and show that de novo ceramide generation through the enzyme ceramide synthase is required. TPA induced rapid ceramide generation, which was detectable by 1 h and increased linearly for 12 h. TPA-induced apoptosis was measurable by 12 h and was progressive for 48 h. Investigations into the mechanism of TPA-induced ceramide generation revealed that acid and neutral sphingomyelinase activities were not enhanced. However, TPA induced an increase in ceramide synthase activity that persisted for at least 16 h. Treatment with fumonisin B1, a specific natural inhibitor of ceramide synthase, abrogated both ceramide production and TPA-induced apoptosis. Ceramide analogues bypassed fumonisin B1 inhibition to initiate apoptosis directly. Thus, ceramide appears to be a necessary signal for TPA-induced apoptosis in LNCaP cells. This represents the first description of a pathway by which PKC may signal apoptosis.
Signaling in and regulation of ionizing radiation-induced apoptosis in endothelial cells.
Exposure of mammalian cells to ionizing radiation leads primarily to DNA damage-induced cell death. The induction of apoptosis by ionizing radiation represents an alternative mode to cell kill. Breakdown of sphingomyelin to produce ceramide by activation of sphingomyelinase is one of the upstream signalling cascades activated in apoptotic cells in response to stimuli such as TNF. Using genetic models of acid sphingomyelinase deficiency, the ceramide generated by radiation-induced activation of sphingomyelinase has been shown to serve as a second messenger in initiating an apoptotic response. PKC activation represents an upstream anti-apoptotic checkpoint at the sphingomyelinase level as well as a checkpoint downstream of ceramide generation. The balance between these pro- and anti-apoptotic systems may determine the magnitude of the observed apoptotic response.
Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation.
The endotoxic shock syndrome is characterized by systemic inflammation, multiple organ damage, circulatory collapse and death. Systemic release of tumor necrosis factor (TNF)-alpha and other cytokines purportedly mediates this process. However, the primary tissue target remains unidentified. The present studies provide evidence that endotoxic shock results from disseminated endothelial apoptosis. Injection of lipopolysaccharide (LPS), and its putative effector TNF-alpha, into C57BL/6 mice induced apoptosis in endothelium of intestine, lung, fat and thymus after 6 h, preceding nonendothelial tissue damage. LPS or TNF-alpha injection was followed within 1 h by tissue generation of the pro-apoptotic lipid ceramide. TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses. Acid sphingomyelinase knockout mice displayed a normal increase in serum TNF-alpha in response to LPS, yet were protected against endothelial apoptosis and animal death, defining a role for ceramide in mediating the endotoxic response. Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha. These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.
Kinase suppressor of Ras is ceramide-activated protein kinase.
A proline-directed serine/threonine ceramide-activated protein (CAP) kinase mediates transmembrane signaling through the sphingomyelin pathway. CAP kinase reportedly initiates proinflammatory TNF alpha action by phosphorylating and activating Raf-1. The present studies delineate kinase suppressor of Ras (KSR), identified genetically in Caenorhabditis elegans and Drosophila, as CAP kinase. Mouse KSR, like CAP kinase, renatures and autophosphorylates as a 100-kDa membrane-bound polypeptide. KSR overexpression constitutively activates Raf-1. TNF alpha or ceramide analogs markedly enhance KSR autophosphorylation and its ability to complex with, phosphorylate, and activate Raf-1. In vitro, low nanomolar concentrations of natural ceramide stimulate KSR to autophosphorylate, and transactivate Raf-1. Other lipid second messengers were ineffective. Moreover, Thr269 the Raf-1 site phosphorylated by CAP kinase, is also recognized by KSR. Thus, by previously established criteria, KSR appears to be CAP kinase.
Stress-induced apoptosis and the sphingomyelin pathway.
The sphingomyelin pathway is a ubiquitous, evolutionarily conserved signaling system initiated by hydrolysis of the plasma membrane phospholipid sphingomyelin to generate the second messenger ceramide. Sphingomyelin degradation is catalyzed by acid and neutral sphingomyelinase (SMase) isoforms. Most, if not all mammalian cells, appear capable of signaling though the sphingomyelin pathway. Diverse receptor types and environmental stresses utilize the sphingomyelin pathway as a downstream effector system. In some cellular systems, ceramide initiates differentiation or cell proliferation, while in other systems, ceramide signals apoptosis. Recent investigations link the activation of neutral SMase to the extracellular signal regulated kinase (ERK) cascade and pro-inflammatory responses, and acid SMase to the stress-activated protein kinase/c-jun kinase (SAPK/JNK) cascade and apoptotic responses. Environmental stresses act directly on membrane to activate acid pH-dependent sphingomyelinase (ASMase), whereas cytokine receptors signal ASMase activation through motifs termed death domains. The present review focuses on mechanisms of activation of ASMase and on ceramide signaling of the apoptotic response.
Inhibition of tumor necrosis factor signal transduction in endothelial cells by dimethylaminopurine.
Tumor necrosis factor (TNF) promotes diverse responses in endothelial cells that are important to the host response to infections and malignancies; however, less is known of the postreceptor events important to TNF action in endothelial cells than in many other cell types. Since phosphorylation cascades are implicated in cytokine signaling, the effects of the protein kinase inhibitor dimethylaminopurine (DMAP) on TNF action in bovine aortic endothelial cells (BAEC) were investigated. In BAEC, TNF promotes phosphorylation of eukaryotic initiation factor 4E (eIF-4E), c-Jun N-terminal kinase (JNK) and ceramide-activated protein kinase activities, Jun-b expression, prostacyclin production, and, when protein synthesis is inhibited, cytotoxicity. DMAP abrogated or significantly attenuated each of these responses to TNF, without affecting the specific binding of TNF to its receptors. Histamine, another agent active in the endothelium, promotes phosphorylation of elongation factor-2 (EF-2) and prostacyclin production, but not phosphorylation of eIF-4E in BAEC. Histamine-stimulated EF-2 phosphorylation was not inhibited and prostacyclin production was unaffected by DMAP. These observations demonstrate that a distinct signal transduction cascade, which can be selectively inhibited by DMAP, promotes the response of BAEC to TNF. Thus, we have identified a reagent, DMAP, that may be useful for characterizing the TNF signal transduction pathway.
Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis.
Stress is believed to activate sphingomyelinase to generate ceramide, which serves as a second messenger in initiating the apoptotic response. Conclusive evidence for this paradigm, however, is lacking. In the present study, we used a genetic approach to address this issue directly. We show that lymphoblasts from Niemann-Pick patients, which have an inherited deficiency of acid sphingomyelinase activity, fail to respond to ionizing radiation with ceramide generation and apoptosis. These abnormalities are reversible up on restoration of acid sphingomyelinase activity by retroviral transfer of human acid sphingomyelinase cDNA. Acid sphingomyelinase knockout mice also expressed defects in radiation-induced ceramide generation and apoptosis in vivo. Comparison with p53 knockout mice revealed that acid sphingomyelinase-mediated apoptosis and p53-mediated apoptosis are likely distinct and independent. These genetic models provide definitive evidence for the involvement of acid sphingomyelinase in one form of stress-induced apoptosis.
Signal transduction through lipid second messengers.
This review emphasizes the generation of glycerolipid and sphingolipid second messengers, and their molecular targets. The role of the phosphatidylinositol transfer protein and phospholipase D in signal transmission, and the structures of the 1, 2-diacylglycerol and calcium-binding sites of protein kinase C are discussed. Further, ceramide signaling through protein kinases and the role of cross-talk in the signaling of apoptosis and inflammation are addressed.
Phosphorylation of Raf by ceramide-activated protein kinase.
The sphingomyelin pathway, initiated by hydrolysis of sphingomyelin to ceramide and stimulation of a Ser/Thr ceramide-activated protein (CAP) kinase, mediates tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta action. CAP kinase is membrane-bound and proline-directed, recognizing the minimal substrate motif Thr-Leu-Pro. TNF may use the sphingomyelin pathway to signal Raf1 to activate the MAP kinase cascade. Evidence shows that cytoplasmic Raf1 binds to GTP-ras upon cellular stimulation, is recruited to the plasma membrane, and activated. How membrane-bound Raf1 is activated is uncertain, but regulation of its kinase activity may involve its phosphorylation. Specific Raf kinases, however, have not hitherto been identified. Here we report that CAP kinase phosphorylates Raf1 on Thr 269, increasing its activity towards MEK (MAP kinase or ERK kinase). Moreover, in intact HL-60 cells, CAP kinase complexes with Raf1 and, in response to TNF and ceramide analogues, phosphorylates and activates Raf1, implicating CAP kinase as a link between the TNF receptor and Raf1.
Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals.
The sphingomyelin pathway, which is initiated by sphingomyelin hydrolysis to generate the second messenger ceramide, signals apoptosis for tumor necrosis factor alpha, Fas, and ionizing radiation. In the present studies, the anticancer drug daunorubicin also stimulated ceramide elevation and apoptosis in P388 and U937 cells. Cell-permeable analogs of ceramide, but not other lipid second messengers, mimicked daunorubicin in inducing apoptosis. Daunorubicin-stimulated ceramide elevation, however, did not result from sphingomyelin hydrolysis, but rather from de novo synthesis via activation of the enzyme ceramide synthase. An obligatory role for ceramide synthase was defined, since its natural specific inhibitor, fumonisin B1, blocked daunorubicin-induced ceramide elevation and apoptosis. These studies demonstrate that ceramide synthase activity can be regulated in eukaryotes and constitute definitive evidence for a requirement for ceramide elevation in the induction of apoptosis.
Ceramide: a signal for apoptosis or mitogenesis?
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Signaling through the sphingomyelin pathway.
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The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling.
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Retinoic acid stimulates the protein kinase C pathway before activation of its beta-nuclear receptor during human teratocarcinoma differentiation.
We previously reported that protein kinase C (PKC) stimulation through phorbol ester (TPA) treatment enhances the effects of all-trans retinoic acid (RA) on immunophenotypic differentiation and RA nuclear receptor (RAR) activation in the multipotential human teratocarcinoma (TC) cell line NTera-2/clone D1 (abbreviated NT2/D1). This study extends prior work in NT2/D1 cells by demonstrating that PKC pathway activation is an early effect of RA treatment which regulates RAR transcriptional activity. RA activated the PKC pathway prior to induction of RAR-beta expression at 6 h, which is an established early marker of RAR activation in NT2/D1 cells. RA caused a transient 1.3-fold increase in intracellular diacylglycerol (DG) at 2 min and a translocation of the gamma isozyme of PKC (PKC-gamma) within 5 min. Transient co-transfection studies provided evidence that PKC pathway activation plays a role in the regulation of RAR-beta expression. In these studies a constitutively active PKC-gamma augmented the RA-mediated transactivation of a luciferase reporter containing the native RAR-beta promoter which has a retinoic-acid-response element (RARE). These findings reveal that PKC pathway activation is an early step in RA-mediated human TC differentiation and that PKC-gamma can potentiate the effects of RA on RAR transcriptional activation.