Increasing Membrane Cholesterol Level Increases the Amyloidogenic Peptide by Enhancing the Expression of Phospholipase C
Figure 4
Transcription inhibitor prevented the effects of augmentation of membrane cholesterol levels. (a) Transcription and translation inhibitors prevented the effect of cholesterol on PLCβ1 and PLCβ3 expressions. APP-transfected HeLa cells were pretreated with the transcription inhibitor, actinomycin-D (Act-D, 10 μM) or translation inhibitor, and cyclohexamide (CHX, 50 μg/mL) for 10 min, which was followed by the additional 0.5 h incubation with 75 μM water-soluble cholesterol. In the presence of Act-D or CHX, the effect of cholesterol on PLCβ1 and PLCβ3 expressions in membrane fractions was prevented. Similar results were obtained from 3 different experiments. β-tubulin was used to confirm the amount of proteins loaded. (b) Transcription inhibitor prevented the effect of cholesterol on PIP2 levels. APP-transfected HeLa cells were incubated in the presence or absence of 10 μM Act-D with 75 μM water-soluble cholesterol for 1 h. PIP2 levels in membrane fractions were measured by using a PIP2 ELISA kit as described in Section 2. In the absence of Act-D (−Act-D), cholesterol decreased PIP2 levels (). However, the presence of Act-D (+Act-D) prevented the effect of cholesterol on PIP2 levels (). (c) Transcription inhibitor prevented the effects of cholesterol on Aβ42 production. APP-transfected HeLa cells were incubated in the presence or absence of 10 μM Act-D with 75 μM water-soluble cholesterol for 4 h. Aβ42 levels were measured from the conditioned media by using ELISA method. In the absence of Act-D (−Act-D), cholesterol increased Aβ42 level (). However, the presence of Act-D (+Act-D) prevented the increase of Aβ42 production induced by cholesterol (). *; **.
