Neutral sphingomyelinase 2 deficiency increases hyaluronan synthesis by up-regulation of Hyaluronan synthase 2 through decreased ceramide production and activation of Akt

Fibroblasts from the fro/fro mouse, with a deletion in the Smpd3 gene coding for the active site of neutral sphingomyelinase 2 (NSMase2), secreted increased amounts of hyaluronan (HA). This was reversed by transfection with the Smpd3 gene, suggesting a connection between sphingolipid and glycosaminoglycan metabolism. The deficiency of NSMase2 resulted in storage of sphingomyelin (SM) and cholesterol with a 50% reduction in ceramides (Cer). RT-PCR and Western blot analysis showed that increased HA secretion resulted from increased hyaluronan synthase 2 (HAS2) activity localized to sphingolipid-enriched lipid rafts. Although cholesterol levels were also elevated in lipid rafts from mouse fibroblasts deficient in lysosomal acid SMase activity (deletion of the Smpd1(-/-) gene), there was no increase in HA secretion. We then showed that in fro/fro fibroblasts, the reduced ceramide was associated with decreased phosphorylation of protein phosphatase 2A (PP2A) and increased phosphorylation of its substrate Akt-p, together with PI3K, PDK1, mTOR (mammalian target of rapamycin), and p70S6K, although PTEN was unaffected. Exogenous ceramide, as well as inhibitors of Akt (Akt inhibitor VIII), PI 3-kinase (LY294002 and wortmannin), and mTOR (rapamycin) reduced secretion of HA, whereas the NSMase2 inhibitor GW4869 increased HA synthesis and secretion. We propose that NSMase2/Cer are the key mediators of the regulation of HA synthesis, via microdomains and the Akt/mTOR pathway.     

Cell surface ceramide controls translocation of transferrin receptor to clathrin-coated pits

Transferrin receptor mediates internalization of transferrin with bound ferric ions through the clathrin-dependent pathway. We found that binding of transferrin to the receptor induced rapid generation of cell surface ceramide which correlated with activation of acid, but not neutral, sphingomyelinase. At the onset of transferrin internalization both ceramide level and acid sphingomyelinase activity returned to their basic levels. Down-regulation of acid sphingomyelinase in cells with imipramine or silencing of the enzyme expression with siRNA stimulated transferrin internalization and inhibited its recycling. In these conditions colocalization of transferrin with clathrin was markedly reduced. Simultaneously, K⁺ depletion of cells which interfered with the assembly of clathrin-coated pits inhibited the uptake of transferrin much less efficiently than it did in control conditions. The down-regulation of acid sphingomyelinase activity led to the translocation of transferrin receptor to the raft fraction of the plasma membrane upon transferrin binding. The data suggest that lack of cell surface ceramide, generated in physiological conditions by acid sphingomyelinase during transferrin binding, enables internalization of transferrin/transferrin receptor complex by clathrin-independent pathway.     

Endoplasmic reticulum stress-mediated inhibition of NSMase2 elevates plasma membrane cholesterol and attenuates NO production in endothelial cells

Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased synthesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intracellular and plasma membrane (PM) cholesterols in BAEC by ~ 3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To confirm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not significantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~ 1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over-expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~ 74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dysfunction as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol.