Acid Sphingomyelinase and Its Redox Amplification in Formation of Lipid Raft Redox Signaling Platforms in Endothelial Cells

Abstract

This study examined the role of acid sphingomyelinase (ASM) and its redox amplification in mediating the formation of lipid raft (LR) redox signaling platforms in coronary arterial endothelial cells (CAECs). Using small interference RNA (siRNA) of ASM, Fas ligand (FasL)-induced increase in ASM activity, production of ceramide, and LR clustering in CAECs were blocked, and clustered Fas was also substantially reduced in detergent-resistant membrane fractions of CAECs. LR clustering, gp91(phox) aggregation, and p47(phox) translocation to the LR clusters induced by FasL were also blocked in ASM-siRNA transfected CAECs. Corresponding to this reduction of LR clustering with NAD(P)H oxidase subunits in ASM-siRNA transfected CAECs, superoxide (O(2)(-*)) production was significantly decreased as measured by either ESR or fluorescent spectrometry. Interestingly, superoxide dismutase (SOD) not only scavenged (O(2)(-*)), but also markedly attenuated LR clustering. Xanthine/xanthine oxidase, an exogenous (O(2)(-*)) generating system, dramatically increased ASM activity and LR clustering in EC membrane and enhanced FasL-induced LR clustering, which were blocked by SOD. These results suggest that that ASM activates LR clustering to form redox signaling platforms, where (O(2)(-*)) production enhances ASM activity, and thereby results in a forwarding amplification of LR and redox signaling. This ASM-mediated feedforwarding mechanism may be critical for an efficient transmembrane signaling through LRs.

Triggering role of acid sphingomyelinase in endothelial lysosome-membrane fusion and dysfunction in coronary arteries

“…The siRNA of ASM gene was purchased from QIAGEN, which has been proven to be efficient to knock down the ASM gene in CAECs by our laboratory's previous studies (20,40) and was further confirmed in the present study (Supplemental Fig. 1).…”

“…One of such signaling platforms was referred to as LR-redox signaling platforms, which was commented to be taking center stage in endothelial cell (EC) redox signaling pathway by editors of Hypertension (18 -20, 40, 41). Over the last 2 yr, we have found that the LR-redox signaling was related to the increased activity of acid sphingomyelinase (ASM), which is a lysosomal glycoprotein and catalyzes the degradation of membrane-bound sphingomyelin into phosphocholine and ceramide (18,19,40). We have also reported that lysosomal vesicles may fuse to the cell membrane and play an important role in the formation of these signaling platforms during death receptor activation (19).…”

Formation and function of ceramide-enriched membrane platforms with CD38 during M1-receptor stimulation in bovine coronary arterial myocytes

“…The activity of ASMase in CAMs was determined as described in our previous studies (39). Briefly, homogenates from CAMs were centrifuged at 1,000 g for 10 min to remove cell nuclei, and 50 l of the supernatant homogenate were used for enzyme activity analysis.…”

Involvement of Ceramide in Cell Death Responses in the Pulmonary Circulation

“…After VEGF receptor blockade, we detected first the activation of the de novo pathway of ceramide synthesis, followed by that of soluble acid SMase, along with increased lung ceramides (26,38). The activation of the acid SMase in endothelial cells, similar to that of neutral SMase in lung epithelial cells (39), may be redoxdependent, as shown both in vivo and in vitro (36,38), and could also be triggered by exogenous ceramide treatment (38). Indeed, direct enrichment of lungs or cells with ceramide itself may induce the acid SMase or the de novo pathway to produce more intracellular ceramides (10, 38), or the soluble SMase to generate paracellular ceramides (26).…”

“…Among these, inflammatory mediators, such as tumor necrosis factor (TNF)-a (10, 30); LPS (31); platelet activating factor (32); ionizing radiation (33); oxidative stress from hypoxia/reoxygenation (34), vascular endothelial growth factor (VEGF) receptor inhibition, or even excess ceramides (35)(36)(37); growth factor deprivation (26); and cigarette smoking (26,38) are particularly relevant to lung diseases that involve pulmonary vascular or microvascular dysfunction. The mechanism of ceramide synthesis may be specific to the stimulus and the context in which it triggers cellular responses.…”