David K. Perry

(1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor of ceramidase

In this study, we have examined the cellular and biochemical activities of the ceramide analog (1S,2R)-Derythro-2-(N-myristoylamino)-1-phenyl-1-propanol (Derythro-MAPP). Addition of 5 μMD-e-MAPP to HL-60 human promyelocytic leukemia cells resulted in a concentration- and time-dependent growth suppression accompanied by an arrest in the G/G phase of the cell cycle; thus mimicking the action of exogenous ceramides. Its enantiomer L-e-MAPP was without effect. Two lines of evidence suggested that D-e-MAPP may not function as a direct analog of ceramide. First, D-e-MAPP possesses a stereochemical configuration opposite to that of D-erythro-ceramide. Second, D-e-MAPP failed to activate ceramide-activated protein phosphatase in vitro. Therefore, we examined if D-e-MAPP functioned indirectly by modulating endogenous ceramide levels. The addition of D-e-MAPP to cells, but not L-e-MAPP, caused a time- and concentration-dependent elevation in endogenous ceramide levels reaching greater than 3-fold over baseline following 24 h of treatment. Both D-e-MAPP and L-e-MAPP underwent similar uptake by HL-60 cells. D-e-MAPP was poorly metabolized, and remained intact in cells, whereas L-e-MAPP underwent a time- and concentration-dependent metabolism; primarily through N-deacylation. In vitro, L-e-MAPP was metabolized by alkaline ceramidase to an extent similar to that seen with C-ceramide. D-e-MAPP was not metabolized. Instead, D-e-MAPP inhibited alkaline ceramidase activity in vitro with an IC of 1-5 μM. D-e-MAPP did not modulate the activity of other ceramide metabolizing enzymes in vitro or in cells, and it was a poor inhibitor of acid ceramidase (IC > 500 μM). Finally, D-e-MAPP inhibited the metabolism of L-e-MAPP in cells. These studies demonstrate that D-e-MAPP functions as an inhibitor of alkaline ceramidase in vitro and in cells resulting in elevation in endogenous levels of ceramide with the consequent biologic effects of growth suppression and cell cycle arrest. These studies point to an important role for ceramidases in the regulation of endogenous levels of ceramide.

Phosphatidic Acid Is a Potent And Selective Inhibitor of Protein Phosphatase 1 and an Inhibitor of Ceramide-mediated Responses

In the present study, we report that phosphatidic acid (PA) functions as a novel, potent, and selective inhibitor of protein phosphatase 1 (PP1). The catalytic subunit of PP1α was inhibited by PA dose-dependently in a noncompetitive manner with a K i value of 80 nm. The inhibition by PA was specific to PP1 as PA failed to inhibit protein phosphatase 2A (PP2A) or PP2B. Furthermore, PA was the most effective and potent inhibitor of PP1 compared with other phospholipids. Because we recently showed that ceramides activated PP1, we next examined the effects of PA on ceramide stimulation of PP1. PA inhibited both basal and ceramide-stimulated PP1 activities, and ceramide showed potent and stereoselective activation of PP1 in the presence of PA. Next, the effects of PA on ceramide-induced responses were examined. Molt-4 cells took up PA dose- and time-dependently such that by 1 and 3 h, uptake of PA was 0.37 and 0.65% of total PA added, respectively. PA at 30 μm and calyculin A at 10 nm (an inhibitor of PP1 and PP2A at low concentrations), but not okadaic acid at 10 nm (a PP2A inhibitor at low concentrations) prevented poly(ADP-ribose) polymerase proteolysis induced by C6-ceramide. Moreover, the combination of PA with okadaic acid prevented retinoblastoma gene product dephosphorylation induced by C6-ceramide. These data suggest that PA functions as a specific regulator of PP1 and may reverse or counteract those effects of ceramide that are mediated by PP1, such as apoptosis and retinoblastoma gene product dephosphorylation.

Ceramide generation by two distinct pathways in tumor necrosis factor α‐induced cell death

Ceramide accumulation in the cell can occur from either hydrolysis of sphingomyelin or by de novo synthesis. In this study, we found that blocking de novo ceramide synthesis significantly inhibits ceramide accumulation and subsequent cell death in response to tumor necrosis factor α. When cells were pre‐treated with glutathione, a proposed cellular regulator of neutral sphingomyelinase, inhibition of ceramide accumulation at early time points was achieved with attenuation of cell death. Inhibition of both pathways achieved near‐complete inhibition of ceramide accumulation and cell death indicating that both pathways of ceramide generation are stimulated. This illustrates the complexity of ceramide generation in cytokine action.

Proteolytic cleavage of phospholipase C-γ1 during apoptosis in Molt-4 cells

Apoptosis is a cell suicide mechanism that requires the activation of cellular death proteases for its induction. We examined whether the progress of apoptosis involves cleavage of phospho‐lipase C‐γΙ (PLC‐γΙ), which plays a pivotal role in mitogenic signaling pathway. Pretreatment of T leu‐kemic Molt‐4 cells with PLC inhibitors such as U‐73122 or ET‐18‐OCH3 potentiated etoposide‐in‐duced apoptosis in these cells. PLC‐γΙ was fragmented when Molt‐4 cells were treated with several apoptotic stimuli such as etoposide, ceramides, and tumor necrosis factor a. Cleavage of PLC‐γΙ was blocked by overexpression of Bcl‐2 and by specific inhibitors of caspases such as Z‐DEVD‐CH2F and YVAD‐cmk. Purified caspase‐3 and caspase‐7, group II caspases, cleaved PLC‐γΙ in vitro and generated a cleavage product of the same size as that observed in vivo, suggesting that PLC‐γΙ is cleaved by group II caspases in vivo. From point mutagenesis studies, Ala‐Glu‐Pro‐Asp 0 was identified to be a cleavage site within PLC‐γΙ. Epidermal growth factor receptor (EGFR)‐induced tyrosine phosphorylation of PLC‐γΙ resulted in resistance to cleavage by caspase‐3 in vitro. Furthermore, cleaved PLC‐γΙ could not be tyrosine‐phosphorylated by EGFR in vitro. In addition, tyrosine‐phosphorylated PLC‐γΙ was not significantly cleaved during etoposide‐in‐duced apoptosis in Molt‐4 cells. This suggests that the growth factor‐induced tyrosine phosphorylation may suppress apoptosis‐induced fragmentation of PLC‐γΙ. We provide evidence for the biochemical relationship between PLC‐γ1‐mediated signal pathway and apoptotic signal pathway, indicating that the defect of PLC‐γ1‐mediated signaling pathway can facilitate an apoptotic progression.—Bae, S. S., Perry, D. K., Oh, Y. S., Choi, J. H., Galadari, S. H., Ghayur, T., Ryu, S. H., Hannun, Y. A., Suh, P.‐G. Proteolytic cleavage of phospholipase C‐γΙ during apoptosis in Molt‐4 cells. FASEB J. 14, 1083–1092 (2000)

Serine palmitoyltransferase: role in apoptotic de novo ceramide synthesis and other stress responses

Serine palmitoyltransferase is the first and rate-limiting enzyme of sphingolipid synthesis. As such, it is a central control point in the synthesis of bioactivate sphingolipids, and it plays an important role in mediating cellular stress responses. In this review, its role in mediating these responses is discussed within the context of de novo ceramide synthesis. Furthermore, a discussion is provided of its regulation as discerned from both yeast and mammalian studies.

The Role of De Novo Ceramide Synthesis in Chemotherapy‐Induced Apoptosis

Abstract: The de novo pathway of sphingolipid synthesis has been implicated as an alternative to sphingomyelinase activation in generating an apoptotic response through ceramide. A chemotherapy agent was used to activate this pathway in a human T‐cell line in order to investigate the role of de novo ceramide synthesis in apoptosis. In data obtained from intact cell radiolabeling studies, it was observed that the first and rate‐limiting enzyme in de novo synthesis, serine palmitoyltransferase, is activated and controls the production of ceramide through this pathway. Furthermore, using agents that selectively inhibit ceramide production by this pathway, partial protection from cell death was observed that was independent of caspase activation. These results reveal that serine palmitoyltransferase, an enzyme that controls sphingolipid synthesis for housekeeping functions, is activated during apoptosis and serves to mediate events in this process.

Proteolytic cleavage of epidermal growth factor receptor by caspases

Apoptotic proteases cleave and inactivate survival signaling molecules such as Akt/PKB, phospholipase C (PLC)‐γ1, and Bcl‐2. We have found that treatment of A431 cells with tumor necrosis factor‐α in the presence of cycloheximide resulted in the cleavage of epidermal growth factor receptor (EGFR) as well as the activation of caspase‐3. Among various caspases, caspase‐1, caspase‐3 and caspase‐7 were most potent in the cleavage of EGFR in vitro. Proteolytic cleavage of EGFR was inhibited by both YVAD‐cmk and DEVD‐fmk in vitro. We also investigated the effect of caspase‐dependent cleavage of EGFR upon the mediation of signals to downstream signaling molecules such as PLC‐γ1. Cleavage of EGFR by caspase‐3 significantly impaired the tyrosine phosphorylation of PLC‐γ1 in vitro. Given these results, we suggest that apoptotic protease specifically cleaves and inactivates EGFR, which plays crucial roles in anti‐apoptotic signaling, to abrogate the activation of EGFR‐dependent downstream survival signaling molecules.

Bcl-2 acts upstream of the PARP protease and prevents its activation.

Apoptosis has recently been extensively studied and multiple factors have been implicated in its regulation. It remains unclear how these factors are ordered in the cell death pathway. Here we investigate the relationship between the inhibitor of apoptosis, bcl-2, and the PARP protease, prlCE/CPP32, recently implicated in apoptosis. Using PARP proteolysis as an indicator of the activation of the PARP protease, we find that the chemotherapeutic agent, etoposide, induces apoptosis and PARP proteolysis in Molt4 cells as early as 4 h with cell death lagging behind this event. In contrast, Molt4 cells that over-express bcl-2 show no PARP proteolysis or cell death. In order to determine if bcl-2 inhibits the PARP protease or its activation, we developed a cell-free system. Using this system with extracts from etoposide-treated cells and purified bovine PARP, we demonostrate that extracts from bcl-2 over-expressing cells cause little or no PARP proteolysis. Whereas, extracts from control vector cells contain an active PARP protease. This protease is inhibited by the tetrapeptide ICE-like protease inhibitor, YVAD-chloromethylketone. Interestingly, this protease is not inhibited by the addition of purified bcl-2 protein. These results rule out that bcl-2 directly inhibits the active protease or that it has an effect downstream of prlCE/CPP32 such as preventing access to the PARP substrate. These results also demonstrate a role of bcl-2 in interfering with an upstream signal required to activate the PARP protease and allow us to begin to order the components in the apoptotic pathway.

Activation of p38 Mitogen-Activated Protein Kinase in Gaucher’s Disease

Gaucher’s disease is caused by defects in acid β-glucosidase 1 (GBA1) and has been also proposed as an inflammatory disease. GBA1 cleaves glucosylceramide to form ceramide, an established bioactive lipid, and defects in GBA1 lead to aberrant accumulation in glucosylceramide and insufficient formation of ceramide. We investigated if the pro-inflammatory kinase p38 is activated in Gaucher’s disease, since ceramide has been proposed to suppress p38 activation. Three Gaucher’s disease mouse models were employed, and p38 was found to be activated in lung and liver tissues of all Gaucher’s disease mice. Most interestingly, neuronopathic Gaucher’s disease type mice, but not non-neuronopathic ones, displayed significant activation of p38 and up-regulation of p38-inducible proinflammatory cytokines in brain tissues. In addition, all type of Gaucher’s disease mice also showed increases in serum IL-6. As cellular signalling is believed to represent an in vivo inflammatory phenotype in Gaucher’s disease, activation of p38 and possibly its-associated formation of proinflammatory cytokines were assessed in fibroblasts established from neuronopathic Gaucher’s disease mice. In mouse Gaucher’s disease cells, p38 activation and IL-6 formation by TNF-α treatment were enhanced as compared to those of wild type. Furthermore, human fibroblasts from Gaucher’s disease patients also displayed increases in p38 activation and IL-6 formation as comparison to healthy counterpart. These results raise the potential that proinflammatory responses such as p38 activation and IL-6 formation are augmented in Gaucher’s disease.