Study On The Role Of NPC1 On SREBP Pathway In Cellular Cholesterol Metabolism

Background and ObjectiveCholesterol is an important lipid in widespread eucaryotic cells and vital for growth and multiplication of cells.Excess cholesterol is toxic to cells because its chemical structure changes to be non-soluble in water.Once cholesterol accumulates along artery walls it can't be mobilized and atherosclerosis even coronary artery disease occurs.As we know,the main content of atherosclerotic plaque is low density lipoprotein(LDL)thus it is very important to investigate cholesterol metabolic pathway,especially LDL,for the prevention and therapy of atherosclerotic heart disease.There are several homeostasis mechanism in normal cells including sterol regulatory element binding protein(SREBP) pathway,cholesterol esterification induced by ACAT and conversion to steroid hormone, bile acid and so on.Many studies show that(niemann-pick C1 protein,NPC1)has been involved in the delivery of LDL to endoplasmic reticulum to be sensed by SREBP cleavage active protein(SCAP)in cholesterol homeostasis.But the precise function of NPC1 remains unknown or even contradictory.The major aim of this thesis is to assess whether NPC1 is involved in the delivery of LDL-cholesterol to the SREBP;SCAP complex in endoplasmic reticulum.This is based on the hypothesis that cholesterol transport to the SREBP;SCAP complex and to ACAT may be distinct processes.The experiments in this thesis will address this by analysing the effects in the absence or presence of LDL-cholesterol on activation of the SREBP pathway in NPC disease.The novel aspect of this thesis is that we will employ more direct measures of SREBP processing than has been reported previously.If NPC1 is involved in LDL-cholesterol delivery to the SREBP;SCAP complex,it may be considered to act at a common point in the pathway of cholesterol delivery to both ACAT and SREBR It will provide more direct evidence to confirm the results in previous studies which infer a link between SREBP activation and ACAT activity in NPC disease.However,if·it is found that LDL-cholesterol delivery to SREBP;SCAP is normal in defective NPC1 cells,this will further confirm that ACAT esterification does not necessarily indicate the amount of cholesterol in the homeostatic pool as sensed by SCAP.It will provide evidence that there are different pathways by which cholesterol moves to ACAT and the SREBP;SCAP complex in the endoplasmic reticulum.A secondary aim will be to further characterise the effect of LDL-cholesterol on Akt phosphorylation in NPC disease.MethodsIn order to study the effect of defective NPC1 on cholesterol metabolism we used two loss of function models.Firstly,pharmacological agents were employed to induce NPC disease.Secondly,mutant cell-lines containing defective NPC1 were utilised to further characterise the effects of LDL in this disease.1.Pharmacological agents;Treating cells with U18666A and progesterone cause accumulation of LDL-cholesterol in the lysosomes and decreased cholesterol esterification,indicating that they can simulate NPC disease.2.Mutant cell-lines;Chinese hamster ovary(CHO)cells have been used to generate NPC1-deficient cells,2-2 and 4-4.In addition,wild-type and NPC1 deficient cells were created with stable expression of PLAP-BP2.3.Tissue culture;Cells were grown as monolayers at 37°C,5%CO2 in various mediums and treatments. 4.Wallac Microbeta luminometer was adopted to determine secreted placental alkaline phosphatase(PLAP)activity expressed as relative light units(RLU)thus the activity of SREBP was differed.5.2μCi of[1-¹⁴C]-oleic acid/1μCi of[1-¹⁴C]-acetate acid was added to all cultural conditions and thin layer chromatography(TLC)was used for cholesterol esterification to verify U18666A does induce NPC disease and cholesterol sythesis assay.6.Filipin staining was utilized to determine the location of cholesterol in cells.7.RNA isolation and gene expression analysis for SREBP target gene,LDL receptor and HMG-CoA reductase,by quantitative real-time PCR(QRT-PCR).8.Analysis of phosphorylated Akt and total Akt by Western Blot to determine if NPC disease has implication for this pathway.9.Fluorescence microscopy was used to locate the movement of SREBP;SCAP complex from endoplasmic reticulum to Golgi.A stable cell-line expressing GFP-SCAP was treated with U18666A and progesterone to determine whether defective NPC1 would affect the localization of SCAP when treated with LDL.10.Statistics;Where error bars are shown on graphs,these represent the means±standard error of the mean(SEM)of separate experiments as indicated in the legends. SPSS 11.0 statistic analysis software was adopted and two-way analysis of variance (ANOVA)was used to test the effect of the presence/absence of LDL, 25-hydroxycholesterol,U18666A,LY295427 and progesterone on PLAP secretion. Two sample student t-test was used to analyse the LDL effect between wild-type, inhibitor and mutants on mRNA expression.Comparisons were performed using the Data analysis function in Microsoft Excel 2003.Data was considered significant if p≤0.05 (two-tailed).Results1.Effect of U18666A on SREBP activation assayA cell-line designated 13A/PS with stable transfection of PLAP-BP2 was treated with U18666A inducing NPC1 phenotype.U18666A does not affect the response of PLAP-BP2 processing to increasing LDL-cholesterol concentrations,while 25-hydroxycholesterol(25HC)served as positive control.Cell cultures treated with U18666A gave similar results to the non-treated cell cultures.This is contrary to the common perception that NPC1 affects SREBP processing and to the results of a transient transfection system using PLAP-BP2 in a previous honours thesis.2.Using LY295427 to test SREBP activation assayLY295427 is able to maintain PLAP-BP2 processing under increasing concentrations of 25HC.In the untreated condition,increasing 25HC was able to reduce PLAP secretion which is reflective of the ability of oxysterols to suppress the SREBP pathway.LY295427-treatment did not block PLAP secretion as much indicating that it was inhibiting the effect of 25HC on the SREBP pathway.These results confirmed that the SREBP activation assay is appropriate.3.Effect of U18666A on cholesterol esterification in the endoplasmic reticulumInhibitors which create the NPC disease phenotype decrease the amount of cholesteryl ester formation under LDL-cholesterol loaded conditions.4.Treatment with U 18666A causes accumulation of LDL-cholesterol in cellsFilipin staining results showed that in the absence of LDL and U18666A cholesterol was located mainly in the plasma membrane.U18666A-treatment without LDL addition caused minor accumulation of cholesterol in what we assume to be lysosomes.In the presence of LDL,cholesterol is located in the plasma membrane and juxtanuclear, perhaps indicating Golgi membrane.When cultured with both U18666A and LDL,there is bright staining in small punctate clusters that is indicative of cholesterol being sequestered in lysosomes of the cell.There is also less staining of cholesterol on the plasma membrane which indicates a problem in delivery of cholesterol to the plasma membrane when cultured with U18666A.5.Repeat of SREBP activation assay using U18666A obtained from Sigma The repeat of this experiment gave a similar trend to the previous experiment in that there was no dampening of the response of SREBP processing to LDL in the presence of U18666A.6.Effect of progesterone on SREBP activation assayIn the absence of LDL,progesterone caused PLAP secretion to decrease.Treatment with LDL further decreased PLAP secretion significantly in the presence or absence of progesterone again confirming the results found using U18666A.25HC was used as a positive control and decreased PLAP secretion with or without progesterone.7.Time-course using 13A/PS cells13A/PS cells were incubated with compactin in the absence or presence of 25HC for time-points.At the earlier time-points up to 8 h,PLAP secretion was similar across for both conditions.It was only at 16 h that there was discrimination between treatments. These results show that shorter time-courses are not appropriate for the PLAP-BP2 assay.8.Creation of an NPC1 mutant cell-line stably expressing PLAP-BP2We tried to create NPC1 mutant cell-line stably expressing PLAP-BP2 and cells were tested in the absence and presence of 25HC,unfortunately none of the mutants expressing PLAP-BP2 gave consistent and clear discrimination in PLAP secretion with and without sterols(data not shown).Due to time constraints this approach was abandoned.9.Expression of mRNA for the LDL receptor,a SREBP-target geneWe employed QRT-PCR to measure the expression of the LDL receptor in CHOK1, 2-2 and 4-4 cell-lines,25HC was used as positive control.Under U18666A treatment condition,Both the drug-induced NPC-diseased phenotype and the NPC1 mutant cell-lines were less responsive to LDL-cholesterol.10.Expression of mRNA for HMG-CoA reductase,a SREBP-target geneSimilar to the LDL receptor,we found using QRT-PCR that the expression of HMG-CoA reductase in response to LDL-cholesterol was blunted in cells with drug-inducing NPC disease and cell-lines containing defective NPC1.11.Functional assay of cholesterol synthesisCHOK1,2-2 and 4-4 cells were incubated with[¹⁴C]-acetate,a precursor for cholesterol synthesis,in the presence or absence of LDL/25HC.The amount of cholesterol synthesized in the wild-type CHO K1 cells decreased by approximately half in response to LDL.Treatment with 25HC reduced cholesterol synthesis to almost zero in all cell-lines.The 2-2 cell-line had the lowest cholesterol synthesis whereas 4-4 cells had the highest.However,both mutant cell-lines showed a blunted response to LDL-cholesterol when compared with the wild-type cells.12.Disturbances in cholesterol homeostasis based on NPC1 have no implications to Akt pathwayA western Blot of phosphorylated Akt and total Akt were performed in CHOK1,2-2 and 4-4 cell-lines in presence or absence of LDL.Akt protein expression is unaffected in NPC1 mutant cells.The results from the densitometry showed that the extent of Akt phosphorylation is only very slightly diminished in NPC1 mutant cell-lines and unaffected by LDL-cholesterol addition in all cell-types.13.Movement of GFP-SCAP under NPC1 drug-induced phenotypeWe analysed the sterol sensing ability under the NPC1 diseased phenotype using immunofluorescence of GFP-SCAP.CHO/pGFP-SCAP cells with stable expression of GFP-SCAP were treated with LDL.In the absence of LDL,cells have a juxtanuclear staining of GFP-SCAP.U18666A causes GFP-SCAP movement to the Golgi in the presence of LDL.Treatment with U18666A maintained Golgi localisation of SCAP up to the 12 h time-point.However,by 24 h this effect had disappeared.This implies that U18666A caused a delay in ability of SCAP to sense LDL cholesterol.Conclusions1.This study shows that functional NPC1 is required for delivery of LDL-cholesterol to SREBP in the endoplasmic reticulum to be sensed to direct processing of SREBP.This sets a precedent in that both LDL-induced cholesterol esterification by ACAT and cholesterol delivery to SREBP;SCAP are paired in NPC1 disease.Hence,it confirms the implications that have assumed ACAT esterification reflects SREBP processing in cells that have defective NPC1.2.Defective NPC1 causes abnormalities in the homeostatic responses to LDL-derived cholesterol in the cell.This thesis shows that defective NPC1 causes a delay in the delivery of LDL-cholesterol to SREBP;SCAP in the endoplasmic reticulum. Therefore it directly shows that functional NPC1 is required for delivery of LDL-cholesterol to SREBP;SCAE3.It also shows that if NPC1 is defective then there is another way for LDL-cholesterol to reach the SREBP;SCAP complex,since LDL is eventually able to reach the endoplasmic reticulum to be sensed.This may occur through the "spill over" from a cholesterol "sink" and/or may indicate the presence of an alternative pathway for LDL-cholesterol delivery which occurs more slowly than the pathway facilitated by NPC1.4.Treatment with oxysterols has been suggested as a possible therapy for patients with defective NPC1 since it seems to restore cholesterol metabolism balance in this disease.This thesis has also shown that oxysterols are able to regulate cholesterol homeostasis in NPC1 mutants and therefore agrees with suggestions that these may form possible designs of treatments for this disease.