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. 2006 May 30;103(22):8408-13.
doi: 10.1073/pnas.0602852103. Epub 2006 May 17.

Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells

Ryan B Corcoran  1 , Matthew P Scott
Affiliations

Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells

Ryan B Corcoran et al. Proc Natl Acad Sci U S A. .

Abstract

Sterol synthesis is required for Sonic hedgehog (Shh) signal transduction. Errors in Shh signal transduction play important roles in the formation of human tumors, including medulloblastoma (MB). It is not clear which products of sterol synthesis are necessary for Shh signal transduction or how they act. Here we show that cholesterol or specific oxysterols are the critical products of sterol synthesis required for Shh pathway signal transduction in MB cells. In MB cells, sterol synthesis inhibitors reduce Shh target gene transcription and block Shh pathway-dependent proliferation. These effects of sterol synthesis inhibitors can be reversed by exogenous cholesterol or specific oxysterols. We also show that certain oxysterols can maximally activate Shh target gene transcription through the Smoothened (Smo) protein as effectively as the known Smo full agonist, SAG. Thus, sterols are required and sufficient for Shh pathway activation. These results suggest that oxysterols may be critical regulators of Smo, and thereby Shh signal transduction. Inhibition of Shh signaling by sterol synthesis inhibitors may offer a novel approach to the treatment of MB and other Shh pathway-dependent human tumors.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Sterol synthesis is required for MB cell proliferation. (A) The SSP. Sites of action of SSPIs are shown by colored bars. Steps catalyzed by enzymes deficient in Smith–Lemli–Opitz syndrome (∗) and desmosterolosis (∗∗) are shown. Multiple enzymatic steps not shown are indicated by double arrows. (B) Sterol synthesis inhibitors block MB cell proliferation. PZp53MED cells were treated with the indicated inhibitors for 72 h, and cell titer was determined. Relative cell titer values represent cell titer of treated cells relative to untreated control. (C) ZGA inhibits proliferation of MB cells selectively compared with normal cell controls (GCP and Hs68). Ptc1−/− fibroblasts, which also show constitutive Shh target gene transcription, were modestly inhibited by ZGA. (D) WSC can restore proliferation in the presence of 10 μM ZGA and 3 μM TPL. (E) Unmodified cholesterol (Chol) and 25-OHC delivered in ethanol vehicle (EtOH) can also restore proliferation in the presence of 10 μM ZGA, whereas 7-OHC cannot.
Fig. 2.
Fig. 2.
Sterol synthesis is required for Shh signaling in MB cells. All gene expression and reporter assays were conducted on confluent cells to ensure that any effect of SSPIs on proliferation did not affect results. Parallel plates were assessed for viable cell number by CellTiter 96 assay to ensure that no cytotoxicity occurred at the drug dosages used. (A) PZp53MED cells treated with 32 μM ZGA for 24 or 48 h show reduced expression of Shh target transcript gli1 relative to untreated controls. Gli1 transcript levels were determined by real-time PCR and normalized to β-actin control. (B) ZGA reduced activity of endogenous Shh-responsive ptc1-lacZ reporter gene in PZp53MED cells relative to untreated controls at 24 and 48 h. (C Upper) ZGA and CPN inhibit MB cell ptc1-lacZ reporter activity with similar maximal efficacy by 48 h. (C Lower) Correlation of IC50s for ptc1-lacZ inhibition (β-gal) and MB cell proliferation (growth) for various SSPIs are shown (NI, no inhibition). (D) WSC can reverse inhibition by 10 μM ZGA and 3 μM TPL of ptc1-lacZ reporter activity in PZp53MED cells (MB) and ptc1−/− fibroblasts (FB) by 48 h. Con, control. (E) 25-OHC, but not 7-OHC, can restore ptc1-lacZ reporter activity in MB cells treated with 10 μM ZGA compared with ethanol vehicle control (EtOH). Values are shown as ratio of reporter activity between ZGA-treated and untreated cells under each condition for 48 h. (F) Constitutive activation of Shh signal transduction by Gli1-transfection maintains proliferation of MB cells in the presence of 10 μM ZGA. Conversely, ZGA completely blocked proliferation in control CFP-transfected cells. Proliferation was assessed by measuring percentage of cells incorporating BrdU after 36 h.
Fig. 3.
Fig. 3.
Sterols directly stimulate Smo activation. (A) Structural similarity between CPN and cholesterol. Standard and three-dimensional structures are shown. Notice the similarity of the four-ring core structure. The eight-carbon side chain is shown in green. Carbons of cholesterol are numbered to indicate the site of hydroxylation in the table in B. (B Left) 25-OHC, but not 7-OHC, can activate ptc1-lacZ expression in PZp53MED cells by 24 h. (B Right) Activities of other compounds tested and their abilities to rescue ptc1-lacZ expression (β-gal) and MB cell proliferation (growth) in the presence of ZGA are shown (NA, no activity). (C) 25-OHC and 20-OHC can activate ptc1-lacZ expression with the same maximal efficacy as the known Smo-binding agonist, SAG. (D) CPN can block activation of ptc1-lacZ reporter activity by 25-OHC. (E and F) Cooperativity of ZGA and CPN for inhibition of ptc1-lacZ expression after 48 h (E) and proliferation of PZp53MED cells after 72 h.
Fig. 4.
Fig. 4.
Model for sterol regulation of Smo activity. (A) Ptc, which is structurally related to transporter and pump proteins, may inhibit Smo by pumping activating sterols away from Smo. In the presence of Shh or in the genetic absence of Ptc, sterols would be free to directly promote Smo activity. (B) Sterols may activate Smo by direct binding. In the absence of sterols, Smo remains in an inactive conformation. When sterols are present, they may bind to Smo, causing an activating conformational change. CPN may compete with sterols for binding to Smo by virtue of the structural similarity of CPN and sterols. Binding of CPN to Smo would stabilize the inactive conformation.
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