ABCMdb

PMID: 25817392

Zarbock R, Kaltenborn E, Frixel S, Wittmann T, Liebisch G, Schmitz G, Griese M
ABCA3 protects alveolar epithelial cells against free cholesterol induced cell death.
Biochim Biophys Acta. 2015 Jul;1851(7):987-95. doi: 10.1016/j.bbalip.2015.03.004. Epub 2015 Mar 25., [PubMed]

Sentences

No. Mutations Sentence Comment

35 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Two clinically relevant mutations belonging to different categories were chosen for this study: p.Q215K leads to mistrafficking [20], while p.E292V causes limited functional impairment of ABCA3 transporter function [21]. Login to comment 42 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Stable transfection of A549 cells with pUB6-ABCA3-WT/Q215K/ E292V vectors was carried out as previously described [22]. Login to comment 86 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Free cholesterol was visualized by filipin staining in A549 cells stably transfected with ABCA3-WT, p.Q215K and p.E292V or mock transfected cells. Login to comment 105 ABCA3 p.Gln215Lys A549 cells expressing wild type ABCA3 (ABCA3-WT) showed significantly reduced levels of FC and of PC compared to mock transfected A549 cells and also compared to cells expressing ABCA3-Q215K (Fig. 1A, D). Login to comment 106 ABCA3 p.Glu292Val Expression of ABCA3-E292V did neither significantly affect FC nor PC levels. Login to comment 107 ABCA3 p.Gln215Lys The expression of both mutations resulted in significantly increased levels of CE compared to both, mock and ABCA3-WT transfected cells (Fig. 1B) Cellular levels of total phospholipids were elevated in ABCA3-Q215K cells compared to ABCA3-WT cells (Fig. 1C). Login to comment 111 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Shown are mRNA levels of SREBPs and INSIGs in stably transfected A549 ABCA3-WT, ABCA3-Q215K and ABCA3-E292V cells and mock transfected cells. Login to comment 118 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys These vesicles are markedly smaller in ABCA3-E292V cells and completely absent in ABCA3-Q215K cells. Login to comment 121 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys This was also the case in cells expressing ABCA3-Q215K and ABCA3-E292V. Login to comment 124 ABCA3 p.Glu292Val In contrast, there was no apparent cholesterol accumulation visible in association with the small ABCA3-positive vesicles observed in cells expressing ABCA3-E292V. Login to comment 135 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Shown are mRNA levels of SREBP target genes in stably transfected A549 ABCA3-WT, ABCA3-Q215K and ABCA3-E292V cells and mock transfected cells. Login to comment 141 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Unlike expression of ABCA3-WT, expression of mutated ABCA3 failed to induce expression of SREBPs and INSIG1; mRNA levels of all three proteins were similar to mock controls in cells expressing either ABCA3-Q215K or ABCA3-E292V (Fig. 4A-C). Login to comment 145 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys While abundant lipid droplets were seen in cells expressing ABCA3-Q215K and ABCA3-E292V and also in mock transfected cells, they were scarcely present in ABCA3-WT expressing cells (Fig. 6). Login to comment 152 ABCA3 p.Glu292Val These vesicles were hardly found in ABCA3-E292V cells where the strongest staining was observed at the plasma membrane. Login to comment 153 ABCA3 p.Gln215Lys In cells expressing ABCA3-Q215K and also in mock transfected cells, a change of cell morphology and membrane blebbing was noticed when compared to untreated control cells, indicating cell death (Fig. 7, arrowheads). Login to comment 155 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys While cell viability was almost unchanged in cells expressing wild type ABCA3 and only a slight reduction was seen in ABCA3 p.E292V and mock transfected cells, viability was reduced significantly in ABCA3-Q215K cells (Fig. 8A). Login to comment 156 ABCA3 p.Gln215Lys In the case of ABCA3-Q215K and mock transfected cells, the effect of cholesterol loading on cell viability was partly reversed when cells were simultaneously treated with U18666A (10 bc;M) which inhibits egress of cholesterol from late endosomes (Fig. 8B). Login to comment 160 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Cells expressing ABCA3-WT, p.Q215K and p.E292V and mock transfected cells were treated with free cholesterol-b2;- methylcyclodextrin complex. Login to comment 165 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Lipid droplets in stably transfected A549 ABCA3-WT, p.Q215K and p.E292V cells and mock transfected cells were visualized by Oil Red O staining and fluorescence microscopy. Login to comment 169 ABCA3 p.Gln215Lys Furthermore, we show that ABCA3 mutations affect the cells' capability to deal with exogenous cholesterol: while in cells expressing wild type ABCA3, FC loading had little effect on cell viability, FC loading proved to be toxic especially to cells expressing the mistrafficking mutation p.Q215K. Login to comment 197 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys (A) Cells expressing ABCA3-WT, p.Q215K and p.E292V and mock transfected cells were treated with free cholesterol-b2;- methylcyclodextrin complex before cell viability was assessed by XTT assay. Login to comment 217 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Here we also delineate differentially the effect of two clinically relevant ABCA3 mutations, i.e. p.Q215K and p.E292V. Login to comment 218 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys While p.Q215K was associated with severe respiratory distress and early death [34], p.E292V seems to permit longer survival [3,13]. Login to comment 219 ABCA3 p.Glu292Val Concordantly, our studies support earlier results that ABCA3-E292V function is moderately impaired [21]. Login to comment 221 ABCA3 p.Gln215Lys In contrast, expression of ABCA3-Q215K resulted in reduced viability not only compared to wild type ABCA3 but also compared to mock transfected cells. Login to comment