Abstract
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.
Highlights
Fibroblasts from the fro/fro mouse contained reduced amounts of ceramides and elevated amounts of hyaluronan (HA)
LC/MS/MS analysis showed that ceramide levels were lower by 50% in fro/fro mouse fibroblasts, calvaria, and brain (Fig. 1E), but were minimally affected in ASMaseϪ/Ϫ fibroblasts and unaffected in ASMaseϪ/Ϫ brain; in contrast SM is greatly elevated in ASMaseϪ/Ϫ tissues
Previous studies showed that multiple cell behaviors including cell proliferation, migration, and apoptosis are influenced by the glycosaminoglycan composition of the extracellular matrix with a major role for HA (29, 49 –52)
Summary
Fibroblasts from the fro/fro mouse contained reduced amounts of ceramides and elevated amounts of hyaluronan (HA). Taking advantage of the fro/fro mouse model, we cultured fibroblasts from ear skin and observed a striking increase in HA synthesis, coupled with significant changes in cell morphology and cell cycle, which were consistent with that in skin fibroblasts from OI patients [25,26,27] This allowed us to further investigate the role of NSMase, and its metabolite the bioactive sphingolipid Cer, in the mechanism of synthesis of HA. We found that increased expression of HAS2, through activation of the PI3K-PDK1Akt-mTOR-p70S6K pathway, was dependent on regulation of the sphingolipid signaling molecule Cer and ceramide-associated protein phosphates 2A (PP2A). This is the first time a connection has been established between sphingolipid and glycosaminoglycan metabolism
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