Defective CFTR increases synthesis and mass of sphingolipids that modulate membrane composition and lipid signaling

Abstract

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that affect protein structure and channel function. CFTR, localized in the apical membrane within cholesterol and sphingomyelin rich regions, is an ABC transporter that functions as a chloride channel. Here, we report that expression of defective CFTR (DeltaF508CFTR or decreased CFTR) in human lung epithelial cell lines increases sphingolipid synthesis and mass of sphinganine, sphingosine, four long-chain saturated ceramide species, C16 dihydroceramide, C22, C24, C26-ceramide, and sphingomyelin, and decreases mass of C18 and unsaturated C18:1 ceramide species. Decreased expression of CFTR is associated with increased expression of long-chain base subunit 1 of serine-palmitoyl CoA, the rate-limiting enzyme of de novo sphingolipid synthesis and increased sphingolipid synthesis. Overexpression of DeltaF508CFTR in bronchoalveolar cells that do not express CFTR increases sphingolipid synthesis and mass, whereas overexpression of wild-type CFTR, but not of an unrelated ABC transporter, ABCA7, decreases sphingolipid synthesis and mass. The data are consistent with a model in which CFTR functions within a feedback system that affects sphingolipid synthesis and in which increased sphingolipid synthesis could reflect a physiological response to sequestration of sphingolipids or altered membrane structure.

Fig. 1.

Expression of ΔF508CFTR and decreased CFTR expression are associated with increased sphingolipid synthesis through recycling and de novo pathways. C38, IB3, 16HBE14o(−) sense, and 16HBE14o(−) antisense cells were incubated for 2 h with ³H-serine to assess sphingolipid synthesis through de novo synthesis or with ³H-sphinganine to assess sphingolipid synthesis through recycling pathways. To measure sphingomyelin synthesis, cells were incubated for 4 h in the presence of ³H-choline. Following lipid extraction and separation by thin-layer chromatography, incorporation of the radioactive tracer into ceramide or sphingomyelin was evaluated by scintillation counting. Sphingolipid synthesis through the de novo pathway is more significantly increased in 16HBE14o(−) antisense cells (P < 0.01) compared with 16HBEo(−) sense control cells than in IB3 cells (P < 0.02) compared with C38 control cells (A). Sphingolipid synthesis though the recycling pathway is equally increased (P < 0.05) in IB3 and 16HBE14o(−) antisense cells compared with their respective controls (B). Sphingomyelin synthesis is equally increased (P < 0.01) in IB3 and 16HBE14o(−) antisense cells compared with their respective controls (C). Asterisks indicate significant difference compared with the respective controls (mean ± SD; * P < 0.05, **P < 0.02, and ***P < 0.01). Data represent the average of at least three experiments carried out in triplicate.

Fig. 2.

Expression of LCB1, the major subunit of the rate-limiting enzyme of de novo sphingolipid synthesis, correlates inversely with expression of CFTR. Expression of CFTR is decreased to ∼10% in 16HBE14o(−) antisense cells compared with expression of CFTR in 16HBE14o(−) sense control cells. LCB1, the major subunit of SPT, the rate-limiting enzyme of sphingolipid de novo synthesis is 1.7-fold (±0.2) higher expressed in 16HBE14o(−) antisense cells than in 16HBE14o(−) sense control cells. Relative expression of LCB1 to CFTR/actin is 0.9 in 16HBE14o(−) sense cells and 16 in 16HBE14o(−) antisense cells (A). IB3 cells express 90% of CFTR expressed by C38 control cells. Expression of LCB1 is 1.3-fold (±0.1) higher in IB3 cells than in C38 controls. Relative expression of LCB1 to CFTR/actin is 0.45 in C38 cells and 0.69 in IB3 cells (B). CFTR was detected with a polyclonal antibody from Cell Signaling.

Fig. 3.

Overexpression of ΔF508 CFTR increases sphingolipid synthesis and LCB1 expression. Overexpression of CFTR decreases sphingolipid synthesis. A: A549 cells, a human bronchioalveolar cell line that does not express CFTR, was transduced with 10¹² pfu/ml AdNull, AdΔF508, or AdCFTR. Forty-eight hours after infection, sphingolipid de novo was assessed by evaluating incorporation of ³H-serine into ceramide. Compared with controls that were transduced with the AdNull control vector, sphingolipid de novo synthesis is increased (P < 0.01) in A549 cells that express ΔF508CFTR and decreased (P < 0.05) in A549 cells that express CFTR. The data represent the average of three different experiments, carried out in triplicate on different days. B: Expression of CFTR and LCB1 was assessed 48 h after infection. A549 cells do not express CFTR (lane 1). Expression of ΔF508CFTR results in expression of a predominant “B band” pattern of CFTR (∼150 kDa) characteristic for ΔF508CFTR and reflecting the trafficking defect. Expression of ΔF508CFTR increases LCB1/actin expression 1.25-fold (±0.2) (lane 2) compared with control (lane 1). Expression of CFTR results in expression of a predominant “C band” pattern of CFTR (∼170 kDa). Expression of CFTR decreases LCB1/actin expression to 0.87 (±0.2) (lane 3) compared with control (lane 1). CFTR was detected with a monoclonal antibody from R and D systems.

Fig. 4.

Overexpression of ABCA7 does not affect LCB1 expression. A549 cells were transfected with pABCA7 or transduced with AdNull or AdΔF508. Protein expression was measured 48 h later. A549 cells do not express ABCA7 (lanes 2, 3). Overexpression of ABCA7 does not increase LCB1 expression [LCB1/actin 0.82 (±0.2), lane 1] compared with controls transduced with AdNull (LCB1/actin expression set to 1, lane 2). Overexpression of AdΔF508CFTR increases LCB1 expression [LCB1/actin expression 1.6 (±0.3) lane 3] compared with controls. Data are representative of more than three experiments that evaluate the effect of pABCA7 on LCB1 expression.

Fig. 5.

Decreased CFTR expression affects composition of ceramide mass. Mass of major cellular saturated ceramide species was determined in 16HBE14o(−) cells that express the sense CFTR construct and 16HBE14o(−) cells transfected with the antisense CFTR construct resulting in decreased expression of CFTR. Compared with controls, mass of C16-dihydroceramide, C22-, C24-, and C26-ceramides is higher (P < 0.05) and mass of C18- and C18:1 ceramide species is lower (P < 0.05) in 16HBE14o(−) cells that express the CFTR antisense construct. Data are expressed as a percentage of change compared with 16HBE14o(−) sense controls.

Fig. 6.

Fenretinide inhibits sphingolipid synthesis and LCB1 expression. A: C38 and IB3 cells were incubated for 6 h in the presence of fenretinide (10 μM), followed by assessment of sphingolipid de novo synthesis, measured by incorporation of ³H-serine into ceramide. After lipid extraction, ceramide was separated by thin-liquid chromatography and counts were measured by scintillation counting. Fenretinide decreases sphingolipid synthesis in IB3 cells. Incubation for 6 h in the presence of fenretinide (10 μM) did not cause apoptosis or cytotoxicity. Asterisks indicate significant difference compared with the respective controls (mean ± SD; * P < 0.05). The data represent average results of three different experiments carried out on different days in triplicate. B: A549 cells were transduced with 10¹² pfu/ml AdCFTR or AdΔF508CFTR. Forty-eight hours later, cells were incubated for 6 h with fenretinide (10 μM) or fumonisin B1 (10 μM). Fifty microliters of total protein was separated on a 7.5% Tris-glycine gel, transferred, and probed for LCB1, CFTR, and actin expression. LCB1 expression is higher in cells that express ΔF508CFTR. Fenretinide and fumonisin decrease LCB1 expression.