Abstract
Hyaluronan synthases (HAS1-3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MbetaCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MbetaCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MbetaCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MbetaCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.
Highlights
Cellular cholesterol content can be modulated by the cholesterol depleting agent methyl--cyclodextrin (MCD)2 [9]
We found that MCD inhibits hyaluronan synthesis but does not change the molecular mass of newly synthesized hyaluronan, whereas it suppressed the mRNA level of HAS2
Our results suggests that cholesterol depletion has a long-term suppressive effect on hyaluronan synthesis
Summary
Cellular cholesterol content can be modulated by the cholesterol depleting agent methyl--cyclodextrin (MCD)2 [9]. Hyaluronan, a large glycosaminoglycan mainly present in the extracellular matrix of vertebrates, is composed of repeating disaccharide units containing glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). It is produced at the plasma membrane by three hyaluronan synthases (HAS1–3). Mammalian cells do not contain cardiolipin in plasma membrane [41], but depletion of cellular cholesterol by MCD suppresses hyaluronan synthesis in aortic smooth muscle cells [42] and the suppression can be reversed by re-addition of cholesterol [42]. Aortic smooth muscle cells from hyperlipidemic rabbits and human skin fibroblasts from hypercholesterolemic patients secrete 2- to 4-fold more hyaluronan to culture medium than normolipidemic controls [42]
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