Methyl-beta-cyclodextrin suppresses hyaluronan synthesis by down-regulation of hyaluronan synthase 2 through inhibition of Akt

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.

FIGURE 1.

MβCD causes cholesterol depletion and suppression of hyaluronan synthesis in MCF-7 cells. A, cells were treated for 4 h with the indicated concentrations of MβCD and cell cholesterol was quantified with the Cholesterol Quantification Kit from Calbiochem. Error bars show the range of two independent experiments. B, different concentrations of MβCD in 1% FBS were added to the cells for 24 h and hyaluronan concentration in the culture medium was analyzed (mean ± S.E. of three independent experiments. In C, hyaluronan was analyzed in cells treated for 24 h with 1 mm MβCD in medium containing 0.5% FBS. Then medium with or without 32 μm cholesterol:MβCD was added for 2 h to refeed the cells with cholesterol after which fresh medium was changed and the cells were allowed to synthesize hyaluronan for 6 h. Means and ranges from two independent experiments are shown. Univariate analysis of variance performed on data in B with three independent experiments showed that the effect of MβCD was significant (p < 0.001), and Tukey's post hoc test indicated that 0.75–2.5 mm MβCD concentrations differed significantly from untreated controls. p < 0.01 for 0.75, 1.0, and 2.5 mm MβCD.

FIGURE 2.

Recovery of hyaluronan synthesis after removal of MβCD from culture medium. MCF-7 cells were treated with 0–2.5 mm MβCD for 24 h. Fresh medium containing 0.5% FBS was added to the cells and changed to fresh medium every 24 h up to 72 h. Hyaluronan accumulation (ng/10,000 cells) to culture medium in 0–24 (A), 24–48 (B), and 48–72 h (C) periods was analyzed by enzyme-linked sorbent assay. Means and ranges from two independent experiments are shown.

FIGURE 3.

Effect of MβCD on the molecular mass of newly synthesized hyaluronan. Cells were labeled with [³H]glucosamine and treated with (gray symbols) or without 2 mm MβCD (black symbols) for 24 h. The molecular mass distributions on the Sephacryl-1000 size exclusion column of ³H-radiolabeled macromolecules susceptible to Streptomyces hyaluronidase digestion, from culture medium (A), trypsin-released material (B), and cell extract (C), are shown. The void volume and total volume of the column were determined by the earliest hyaluronan-positive fractions of standard hyaluronan (Hyalose, 2,500 kDa) and the elution position of glucuronic acid, respectively. The peak elution positions of hyaluronan standards (2,500, 500, and 150 kDa) are indicated. The two molecular mass profiles for untreated and treated cells come from two parallel wells. The experiment was repeated twice with similar results.

FIGURE 4.

Effect of MβCD on the cellular contents of UDP-GlcUA and UDP-GlcNAc. Cells were treated with 0–2.5 mm MβCD for 24 h, after which UDP-sugars were extracted with acetonitrile and the levels of UDP-GlcUA and UDP-GlcNAc analyzed by anion exchange HPLC with a CarboPac^TM PA1 column. The data represent mean ± S.E. from four experiments. Univariate analysis of variance indicated that MβCD had a statistically significant effect on both UDP-GlcUA and UDP-GlcNAc levels (p < 0.05). However, comparing the individual means to untreated controls with Tukey's post hoc test indicated significant differences in just the UDP-GlcNAc level when using 1 and 2.5 mm MβCD (p < 0.05).

FIGURE 5.

Effect of MβCD on HAS2 and HAS3 mRNA levels. Cells were treated with the indicated concentrations of MβCD for 2, 4, and 24 h, and HAS2 (A–C) and HAS3 (D–F) mRNA levels (HAS1 was not expressed) were analyzed with quantitative RT-PCR. ARP0 was used as a control gene. The data represent means and ranges from two to three independent experiments. Analysis of variance (univariate analysis of variance) was performed for the data generated from three independent experiments (B and D), allowing the statistical analysis. In B the effect of MβCD was highly significant (p < 0.001). Tukey's post hoc test, comparing the individual values to untreated controls, showed that all MβCD concentrations caused a significant decrease in HAS2 expression. p < 0.01 for 0.5 mm and p < 0.001 for 0.75, 1.0, and 2.5 mm MβCD.

FIGURE 6.

Effect of signaling inhibitors on hyaluronan synthesis and HAS2 mRNA level. Hyaluronan concentration in the medium of MCF-7 cells was treated for 24 h with 1 μm wortmannin (PI3K inhibitor), 25 μm LY294002 (PI3K inhibitor), 1 μm Akt inhibitor VIII, 100 nm rapamycin (mTOR inhibitor), 2 μm PD98059 (MEK inhibitor), 10 mm Y-27632 (ROCK inhibitor), 1 mm 14-22 (PKA inhibitor), 8 μm BIM (PKC inhibitor), 50 nm toxin B (small GTPase inhibitor), 100 μm Rac1 inhibitor NSC23766, 1 mm LiCl (GSK3β inhibitor), 200 nm Compound C (adenosine monophosphate kinase inhibitor), and 100 nm PP2 (Src inhibitor) for 24 h (A). The data represent means and ranges from two to four independent experiments. For the HAS2 expression analysis, cells were incubated with PI3K inhibitors wortmannin (1 μm) and LY294002 (25 μm) and the Akt inhibitor VIII (1 μm) for 4 h and the HAS2 mRNA level was analyzed with quantitative RT-PCR (B).

FIGURE 7.

Effect of cholesterol depletion on Akt and p70S6k activation. Phosphorylation states of Akt (T308, S473) (A) and p70S6k (Thr-229, Thr-389, Thr-421/Ser-424) (B) were analyzed with the human Phospho-Kinase Array Kit in cells treated with or without 1 mm MβCD for 10 min, 30 min, 2 h, and 4 h. The data represent means of fold-changes of phosphorylation between untreated and MβCD-treated samples. The experiments were performed twice at 10 min, 30 min, and 4 h and once at the 2-h time point. Phosphorylation of cytosolic Akt was analyzed with Western blotting after a 4-h treatment with 1 mm MβCD (C).

FIGURE 8.

Schematic presentation of MβCD-induced signaling leading to down-regulation of HAS2 and hyaluronan synthesis in MCF-7 cells. In the figure, GR and RE represent growth factor receptor and response element, respectively.