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Lysosomal Acid Lipase Deficiency Impairs Regulation of ABCA1 Gene and Formation of High Density Lipoproteins in Cholesteryl Ester Storage Disease*

Open AccessPublished:July 10, 2011DOI:https://doi.org/10.1074/jbc.M111.274381
      ATP-binding cassette transporter A1 (ABCA1) mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, and its expression is regulated primarily by oxysterol-dependent activation of liver X receptors. We previously reported that ABCA1 expression and HDL formation are impaired in the lysosomal cholesterol storage disorder Niemann-Pick disease type C1 and that plasma HDL-C is low in the majority of Niemann-Pick disease type C patients. Here, we show that ABCA1 regulation and activity are also impaired in cholesteryl ester storage disease (CESD), caused by mutations in the LIPA gene that result in less than 5% of normal lysosomal acid lipase (LAL) activity. Fibroblasts from patients with CESD showed impaired up-regulation of ABCA1 in response to low density lipoprotein (LDL) loading, reduced phospholipid and cholesterol efflux to apolipoprotein A-I, and reduced α-HDL particle formation. Treatment of normal fibroblasts with chloroquine to inhibit LAL activity reduced ABCA1 expression and activity, similar to that of CESD cells. Liver X receptor agonist treatment of CESD cells corrected ABCA1 expression but failed to correct LDL cholesteryl ester hydrolysis and cholesterol efflux to apoA-I. LDL-induced production of 27-hydroxycholesterol was reduced in CESD compared with normal fibroblasts. Treatment with conditioned medium containing LAL from normal fibroblasts or with recombinant human LAL rescued ABCA1 expression, apoA-I-mediated cholesterol efflux, HDL particle formation, and production of 27-hydroxycholesterol by CESD cells. These results provide further evidence that the rate of release of cholesterol from late endosomes/lysosomes is a critical regulator of ABCA1 expression and activity, and an explanation for the hypoalphalipoproteinemia seen in CESD patients.

      Introduction

      High density lipoproteins (HDL) are thought to protect against atherosclerosis by diverse mechanisms, including mediating reverse cholesterol transport and anti-inflammatory effects (
      • Francis G.A.
      ). The rate-limiting step in HDL particle formation is the lipidation of apolipoprotein A-I (apoA-I) and other HDL apolipoproteins by the membrane lipid transporter ATP-binding cassette transporter A1 (ABCA1) (
      • Oram J.F.
      • Heinecke J.W.
      ). Expression of ABCA1 is induced by increased cell cholesterol content, through oxysterol-dependent activation of the nuclear liver X receptors (LXRs)
      The abbreviations used are: LXR
      liver X receptor
      CESD
      cholesteryl ester storage disease
      27-HC
      27-hydroxycholesterol
      LAL
      lysosomal acid lipase
      LPDS
      lipoprotein-deficient serum
      NPC
      Niemann-Pick type C
      LPDS
      lipoprotein-deficient serum
      rh
      recombinant human.
      on the promoter region of ABCA1 (
      • Costet P.
      • Luo Y.
      • Wang N.
      • Tall A.R.
      ,
      • Venkateswaran A.
      • Laffitte B.A.
      • Joseph S.B.
      • Mak P.A.
      • Wilpitz D.C.
      • Edwards P.A.
      • Tontonoz P.
      ). Further cholesterol efflux to HDL can be mediated via ABCG1, also regulated transcriptionally by LXR (
      • Kennedy M.A.
      • Venkateswaran A.
      • Tarr P.T.
      • Xenarios I.
      • Kudoh J.
      • Shimizu N.
      • Edwards P.A.
      ,
      • Wang N.
      • Lan D.
      • Chen W.
      • Matsuura F.
      • Tall A.R.
      ). However, the roles of different intracellular cholesterol pools, including de novo synthesized cholesterol, late endosomal/lysosomal cholesterol, and plasma membrane cholesterol, in the regulation of oxysterol production and ABCA1 expression are poorly understood.
      We have previously demonstrated that regulation of ABCA1 is impaired in the lysosomal cholesterol storage disorder Niemann-Pick disease type C1 (NPC1), leading to reduced HDL particle formation by human NPC1 disease fibroblasts (
      • Choi H.Y.
      • Karten B.
      • Chan T.
      • Vance J.E.
      • Greer W.L.
      • Heidenreich R.A.
      • Garver W.S.
      • Francis G.A.
      ). The defect in the lipidation of apoA-I was overcome by addition of an exogenous non-oxysterol synthetic ligand for LXR to up-regulate ABCA1, suggesting ABCA1 might be able to mobilize cholesterol from late endosomes/lysosomes even in the presence of NPC1 deficiency (
      • Boadu E.
      • Choi H.Y.
      • Lee D.W.
      • Waddington E.I.
      • Chan T.
      • Asztalos B.
      • Vance J.E.
      • Chan A.
      • Castro G.
      • Francis G.A.
      ). We also determined that plasma HDL-C was low in 17/21 patients studied with NPC disease (
      • Choi H.Y.
      • Karten B.
      • Chan T.
      • Vance J.E.
      • Greer W.L.
      • Heidenreich R.A.
      • Garver W.S.
      • Francis G.A.
      ), further confirming a recent report of a larger cohort of NPC1 subjects (
      • Garver W.S.
      • Jelinek D.
      • Meaney F.J.
      • Flynn J.
      • Pettit K.M.
      • Shepherd G.
      • Heidenreich R.A.
      • Vockley C.M.
      • Castro G.
      • Francis G.A.
      ). Low plasma HDL-C in patients with NPC1 disease occurs independent of their plasma triglyceride levels (
      • Garver W.S.
      • Jelinek D.
      • Meaney F.J.
      • Flynn J.
      • Pettit K.M.
      • Shepherd G.
      • Heidenreich R.A.
      • Vockley C.M.
      • Castro G.
      • Francis G.A.
      ), further suggesting that impaired ABCA1 regulation as a consequence of reduced flux of cholesterol out of lysosomes is the cause of the hypoalphalipoproteinemia in this disease.
      In this study, we have extended these findings to another lysosomal cholesterol storage disorder, cholesteryl ester storage disease (CESD), caused by deficiency of lysosomal acid lipase (LAL) activity (
      • Assmann G.
      • Seedorf U.
      ). CESD and its more severe form Wolman disease are autosomal recessive diseases caused by mutations in the LIPA gene that encodes for LAL, the sole enzyme responsible for acidic hydrolysis of cholesteryl esters and triglycerides delivered from lipoproteins to lysosomes (
      • Goldstein J.L.
      • Dana S.E.
      • Faust J.R.
      • Beaudet A.L.
      • Brown M.S.
      ,
      • Du H.
      • Sheriff S.
      • Bezerra J.
      • Leonova T.
      • Grabowski G.A.
      ). In contrast to Wolman disease, in which complete absence of LAL activity results in death usually in the first 6 months of life, splice mutations of LIPA that result in the 5–10% residual LAL activity in CESD allow patients to survive usually to adulthood. In addition to hepatosplenomegaly, individuals with CESD exhibit premature atherosclerosis and plasma HDL-C levels that are approximately half the normal levels (
      • Assmann G.
      • Seedorf U.
      ). The reason for low HDL-C in CESD has not previously been known. Here, we describe impaired regulation of ABCA1 in human CESD fibroblasts, reduced lipid efflux to apoA-I and HDL particle formation, induction of the same defects by inhibition of LAL activity in normal fibroblasts, and correction of ABCA1 expression and HDL particle formation in CESD cells treated with LAL-containing conditioned medium from normal fibroblasts or recombinant human LAL. We also demonstrate impaired oxysterol generation in CESD fibroblasts, as previously shown in NPC1−/− and NPC2−/− fibroblasts (
      • Frolov A.
      • Zielinski S.E.
      • Crowley J.R.
      • Dudley-Rucker N.
      • Schaffer J.E.
      • Ory D.S.
      ), and correction of oxysterol formation in CESD cells following incubation with LAL-containing conditioned medium or purified LAL enzyme. Together with our findings in NPC1 disease, these results provide a likely reason for the low HDL-C in CESD-impaired regulation of ABCA1, and they further demonstrate the critical importance of the rate of flux of cholesterol out of lysosomes in the regulation of ABCA1 expression and HDL particle formation.

      DISCUSSION

      The studies presented here provide several additional lines of evidence that the rate of flux of cholesterol out of lysosomes is a key regulator of ABCA1 expression and activity and therefore HDL particle formation. Human CESD fibroblasts with very low residual activity of LAL and slowed rate of hydrolysis of cholesteryl esters in late endosome/lysosomes exhibited a marked decrease in LDL-stimulated expression of ABCA1 at the mRNA and protein level, resulting in impaired ABCA1-dependent phospholipid and cholesterol efflux to apoA-I, and reduced production of larger α-HDL particles. Treatment of normal fibroblasts with chloroquine to inhibit LAL activity induced the same defects in LDL-stimulated increases in ABCA1 expression and cholesterol efflux to apoA-I as seen in the CESD cells. Treatment of CESD cells with LAL-containing conditioned medium from normal cells or purified recombinant human LAL corrected cholesteryl ester hydrolysis, ABCA1 expression, cholesterol efflux to apoA-I, and formation of α-HDL particles to levels similar to those seen in normal fibroblasts. Formation of the key oxysterol formed in response to LDL loading in fibroblasts, 27-hydroxycholesterol, was reduced in CESD fibroblasts, consistent with reduced ABCG1 as well ABCA1 expression in these cells. 27-HC formation was increased upon addition of LAL-containing medium to correct cholesteryl ester hydrolysis. Together with our previous findings using cells from patients with the lysosomal cholesterol storage disorder NPC1 disease (
      • Choi H.Y.
      • Karten B.
      • Chan T.
      • Vance J.E.
      • Greer W.L.
      • Heidenreich R.A.
      • Garver W.S.
      • Francis G.A.
      ,
      • Boadu E.
      • Choi H.Y.
      • Lee D.W.
      • Waddington E.I.
      • Chan T.
      • Asztalos B.
      • Vance J.E.
      • Chan A.
      • Castro G.
      • Francis G.A.
      ), these results demonstrate further the critical role of the rate of flux of cholesterol out of late endosomes/lysosomes in regulating ABCA1 expression and HDL particle formation. They also provide the first likely explanation for the low plasma HDL-C seen in CESD patients, impaired regulation of ABCA1.
      In both NPC1 disease and CESD, previous studies using cultured fibroblasts showed that the reduced rate of release of unesterified cholesterol from late endosomes/lysosomes leads to impaired down-regulation of HMG-CoA reductase and LDL receptor activity and therefore inappropriately high levels of de novo cholesterol synthesis and LDL uptake (
      • Goldstein J.L.
      • Dana S.E.
      • Faust J.R.
      • Beaudet A.L.
      • Brown M.S.
      ,
      • Pentchev P.G.
      • Comly M.E.
      • Kruth H.S.
      • Tokoro T.
      • Butler J.
      • Sokol J.
      • Filling-Katz M.
      • Quirk J.M.
      • Marshall D.C.
      • Patel S.
      • et al.
      ). Reduced trafficking of unesterified cholesterol to the endoplasmic reticulum also results in reduced levels of cholesterol esterification by acyl-CoA:cholesterol acyltransferase in both diseases (
      • Goldstein J.L.
      • Dana S.E.
      • Faust J.R.
      • Beaudet A.L.
      • Brown M.S.
      ,
      • Pentchev P.G.
      • Comly M.E.
      • Kruth H.S.
      • Vanier M.T.
      • Wenger D.A.
      • Patel S.
      • Brady R.O.
      ). At the same time, our previous work (
      • Choi H.Y.
      • Karten B.
      • Chan T.
      • Vance J.E.
      • Greer W.L.
      • Heidenreich R.A.
      • Garver W.S.
      • Francis G.A.
      ,
      • Boadu E.
      • Choi H.Y.
      • Lee D.W.
      • Waddington E.I.
      • Chan T.
      • Asztalos B.
      • Vance J.E.
      • Chan A.
      • Castro G.
      • Francis G.A.
      ) and this study suggest the low rate of cholesterol egress from lysosomes and oxysterol generation in both these diseases results in reduced ABCA1 expression and HDL particle formation. Fibroblasts from NPC1 and CESD patients therefore fail to sense the accumulation of excess cholesterol in late endosomes/lysosomes and to up-regulate ABCA1 appropriately in response to cholesterol loading, thereby resulting in impaired HDL formation.
      27-HC has been shown to be the predominant oxysterol formed and primary regulator of both HMG-CoA reductase and ABCA1 expression in response to LDL or acetylated LDL loading in human fibroblasts and other cell types (
      • Axelson M.
      • Larsson O.
      ,
      • Fu X.
      • Menke J.G.
      • Chen Y.
      • Zhou G.
      • MacNaul K.L.
      • Wright S.D.
      • Sparrow C.P.
      • Lund E.G.
      ). The reduced generation of 27-HC in NPC disease (
      • Frolov A.
      • Zielinski S.E.
      • Crowley J.R.
      • Dudley-Rucker N.
      • Schaffer J.E.
      • Ory D.S.
      ) and in CESD cells in response to LDL loading found in this study is striking also in demonstrating the key role of lysosomally derived cholesterol in the formation of this key regulatory oxysterol required for ABCA1 expression. Despite increased HMG-CoA reductase activity and de novo cholesterol synthesis in both CESD and NPC disease cells (
      • Goldstein J.L.
      • Dana S.E.
      • Faust J.R.
      • Beaudet A.L.
      • Brown M.S.
      ,
      • Pentchev P.G.
      • Comly M.E.
      • Kruth H.S.
      • Tokoro T.
      • Butler J.
      • Sokol J.
      • Filling-Katz M.
      • Quirk J.M.
      • Marshall D.C.
      • Patel S.
      • et al.
      ), newly synthesized cholesterol is apparently not contributing a significant pool of cholesterol or regulatory oxysterols affecting ABCA1 expression. Although synthesis of 24(S),25-epoxycholesterol has been shown to increase coordinately with de novo cholesterol synthesis via HMG-CoA reductase (
      • Wong J.
      • Quinn C.M.
      • Brown A.J.
      ), synthesis of this oxysterol is apparently not enough to impact ABCA1 expression in the face of reduced flux of cholesterol out of lysosomes. Further demonstration of the specific and critical role of lysosomally derived cholesterol in regulating ABCA1 expression was seen in our experiments using chloroquine, where levels of ABCA1 mRNA were reduced to near zero in cells pretreated with lipoprotein-deficient serum or following LDL loading (Fig. 3A).
      Treatment of CESD cells with the non-oxysterol LXR agonist TO-901317 increased ABCA1 mRNA and protein levels up to either normal or higher levels than those seen in untreated normal fibroblasts (Fig. 4, A and B). In contrast to our previous findings of complete correction of apoA-I-mediated cholesterol efflux and HDL particle formation in NPC1-deficient fibroblasts treated with this LXR agonist (
      • Boadu E.
      • Choi H.Y.
      • Lee D.W.
      • Waddington E.I.
      • Chan T.
      • Asztalos B.
      • Vance J.E.
      • Chan A.
      • Castro G.
      • Francis G.A.
      ), CESD cells showed a persistent reduction in apoA-I-mediated cholesterol efflux (Fig. 4C). These results suggest that treatment with this agonist and increased expression of ABCA1 can bypass a deficiency in NPC1 activity to induce mobilization of lysosomal (unesterified) cholesterol, but it cannot overcome the deficiency in LAL activity and reduced LDL cholesteryl ester hydrolysis in CESD cells. These results also support the conclusion of previous studies (
      • Chen W.
      • Sun Y.
      • Welch C.
      • Gorelik A.
      • Leventhal A.R.
      • Tabas I.
      • Tall A.R.
      ,
      • Chen W.
      • Wang N.
      • Tall A.R.
      ) that lysosomally derived cholesterol forms a significant fraction of the substrate pool of cholesterol mobilized by ABCA1 for HDL particle formation.
      Our findings that addition of LAL-containing conditioned medium from normal fibroblasts or purified LAL rescued ABCA1 expression and activity are consistent with previous results showing correction of LDL cholesteryl ester hydrolysis and new cholesteryl ester formation as well as suppression of HMG-CoA reductase by addition of normal fibroblast conditioned medium to CESD fibroblasts (
      • Brown M.S.
      • Sobhani M.K.
      • Brunschede G.Y.
      • Goldstein J.L.
      ). Correction of LDL-derived radiolabeled cholesteryl ester and unesterified cholesterol levels in CESD cells to the same levels as seen in normal fibroblasts (Fig. 5, D and E, and supplemental Fig. 1, C and D) indicated LAL in the rescue medium was targeted to and active in late endosomes/lysosomes of the CESD cells. Correction of LAL activity also resulted in normalization of apoA-I-mediated cholesterol efflux (Fig. 5C and supplemental Fig. 1B), HDL particle formation (Fig. 6 and supplemental Fig. 2), and 27-HC formation (Fig. 7 and supplemental Fig. 3) in the CESD cells. Although differences in 27-HC production between normal and CESD cells in the absence or presence of purified LAL enzyme did not reach significance, the increases in 27-HC production by CESD cells treated with both conditioned medium or purified LAL were sufficient to correct ABCA1 expression and activity in both cases. These results suggest that the rate of production of 27-HC, in addition to total 27-HC mass produced over a 24-h period, is important in correcting ABCA1 expression following normalization of cholesteryl ester hydrolysis. This increase in 27-HC formation and correction of the regulatory defect in ABCA1 expression and activity by adding back LAL to CESD cells provides further evidence for reduced lysosomal cholesteryl ester hydrolysis and flux of unesterified cholesterol out of the late endosome/lysosome compartment, and therefore the reduced delivery of this cholesterol to sites of oxysterol generation, as the reason for impaired ABCA1 regulation and HDL formation in CESD.
      A striking aspect of both NPC disease and CESD is the low plasma HDL-C in humans with these disorders (
      • Garver W.S.
      • Jelinek D.
      • Meaney F.J.
      • Flynn J.
      • Pettit K.M.
      • Shepherd G.
      • Heidenreich R.A.
      • Vockley C.M.
      • Castro G.
      • Francis G.A.
      ,
      • Assmann G.
      • Seedorf U.
      ) but not in the mouse models of NPC1 and LAL deficiency (
      • Xie C.
      • Turley S.D.
      • Dietschy J.M.
      ,
      • Du H.
      • Heur M.
      • Duanmu M.
      • Grabowski G.A.
      • Hui D.Y.
      • Witte D.P.
      • Mishra J.
      ). Previous studies indicated hepatic ABCA1 is a critical regulator of plasma HDL-C in mice (
      • Timmins J.M.
      • Lee J.Y.
      • Boudyguina E.
      • Kluckman K.D.
      • Brunham L.R.
      • Mulya A.
      • Gebre A.K.
      • Coutinho J.M.
      • Colvin P.L.
      • Smith T.L.
      • Hayden M.R.
      • Maeda N.
      • Parks J.S.
      ) and that hepatocyte ABCA1 is not reduced in NPC1−/− mice (
      • Wang M.D.
      • Franklin V.
      • Sundaram M.
      • Kiss R.S.
      • Ho K.
      • Gallant M.
      • Marcel Y.L.
      ). The absence of low plasma HDL-C in LAL-deficient mice is consistent with these observations, because mouse hepatic ABCA1 is reported to be unresponsive to LXR stimulation (
      • Brunham L.R.
      • Kruit J.K.
      • Pape T.D.
      • Parks J.S.
      • Kuipers F.
      • Hayden M.R.
      ,
      • Tang W.
      • Ma Y.
      • Jia L.
      • Ioannou Y.A.
      • Davies J.P.
      • Yu L.
      ) and would not be expected to show reduced expression in the face of reduced flux of cholesterol out of lysosomes and oxysterol generation with LAL deficiency. Human hepatocyte ABCA1, conversely, is responsive to LXR activation in both primary human hepatocytes (
      • Whitney K.D.
      • Watson M.A.
      • Goodwin B.
      • Galardi C.M.
      • Maglich J.M.
      • Wilson J.G.
      • Willson T.M.
      • Collins J.L.
      • Kliewer S.A.
      ,
      • Menke J.G.
      • Macnaul K.L.
      • Hayes N.S.
      • Baffic J.
      • Chao Y.S.
      • Elbrecht A.
      • Kelly L.J.
      • Lam M.H.
      • Schmidt A.
      • Sahoo S.
      • Wang J.
      • Wright S.D.
      • Xin P.
      • Zhou G.
      • Moller D.E.
      • Sparrow C.P.
      ) and HepG2 cells (
      • Aravindhan K.
      • Webb C.L.
      • Jaye M.
      • Ghosh A.
      • Willette R.N.
      • DiNardo N.J.
      • Jucker B.M.
      ,
      • Zelcer N.
      • Hong C.
      • Boyadjian R.
      • Tontonoz P.
      ). Human hepatocyte ABCA1 would therefore be expected to show reduced expression in the face of reduced LAL (and NPC1) activity and the reduced production of oxysterol agonist of LXR, which we have demonstrated here in fibroblasts. These conclusions are consistent with both a major role of hepatic ABCA1 in predicting plasma HDL-C and the approximately half-normal HDL-C levels seen in humans with CESD (
      • Assmann G.
      • Seedorf U.
      ,
      • Ginsberg H.N.
      • Le N.A.
      • Short M.P.
      • Ramakrishnan R.
      • Desnick R.J.
      ). Additional studies are required to explore the role of LAL or NPC1 deficiency on human hepatocyte ABCA1 expression and HDL formation.
      In summary, the results presented provide further evidence that the rate of flux of cholesterol out of late endosomes/lysosomes is a critical regulator of the expression of ABCA1 and HDL particle formation, and is not corrected by the increased de novo cholesterol synthesis seen in cells from patients with two different diseases of lysosomal cholesterol storage, cholesteryl ester storage disease and NPC1 disease. These results also provide the first plausible explanation for the hypoalphalipoproteinemia seen in CESD, impaired regulation of ABCA1. We therefore propose a model for CESD cells where hydrolysis of cholesteryl esters from endocytosed LDL in late endosomes and lysosomes is impaired, and there is a reduced rate of unesterified cholesterol release from these compartments to other intracellular sites for regulatory effects, including production of 27-HC, and therefore reduced LXR-dependent regulation of ABCA1. In addition, less unesterified cholesterol is available to join the substrate pool of ABCA1 for new HDL particle formation. Although we did not find an up-regulation of ABCA1 expression in normal cells in response to addition of conditioned medium containing LAL, further studies are required to assess the potential role of LAL as a target to increase ABCA1 expression and HDL formation at the cellular and clinical level.

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