Oxidized Lipoproteins Inhibit Surfactant Phosphatidylcholine Synthesis via Calpain-mediated Cleavage of CTP:Phosphocholine Cytidylyltransferase
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m304316200
ISSN1083-351X
AutoresJiming Zhou, Alan J. Ryan, Jheem D. Medh, Rama K. Mallampalli,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoWe investigated effects of pro-atherogenic oxidized lipoproteins on phosphatidylcholine (PtdCho) biosynthesis in murine lung epithelial cells (MLE-12). Cells surface-bound, internalized, and degraded oxidized low density lipoproteins (Ox-LDL). Ox-LDL significantly reduced [3H]choline incorporation into PtdCho in cells by selectively inhibiting the activity of the rate-regulatory enzyme, CTP:phosphocholine cytdylyltransferase (CCT). Ox-LDL coordinately increased the cellular turnover of CCTα protein as determined by [35S]methionine pulse-chase studies by inducing the calcium-activated proteinase, calpain. Forced expression of calpain or exposure of cells to the calcium ionophore, A23187, increased CCTα degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCTα degradation. The effects of Ox-LDL on CCTα breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCTα isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCTα primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCTα generated p26, which upon NH2-terminal sequencing localized a calpain attack site within the CCTα amino terminus. Expression of a CCTα mutant where the amino-terminal cleavage site and a putative carboxyl-terminal hydrolysis region were modified resulted in an enzyme that was significantly less sensitive to proteolytic cleavage and restored the ability of cells to synthesize surfactant PtdCho after Ox-LDL treatment. Thus, these results provide a critical link between proatherogenic lipoproteins and their metabolic target, CCTα, resulting in impaired surfactant metabolism. We investigated effects of pro-atherogenic oxidized lipoproteins on phosphatidylcholine (PtdCho) biosynthesis in murine lung epithelial cells (MLE-12). Cells surface-bound, internalized, and degraded oxidized low density lipoproteins (Ox-LDL). Ox-LDL significantly reduced [3H]choline incorporation into PtdCho in cells by selectively inhibiting the activity of the rate-regulatory enzyme, CTP:phosphocholine cytdylyltransferase (CCT). Ox-LDL coordinately increased the cellular turnover of CCTα protein as determined by [35S]methionine pulse-chase studies by inducing the calcium-activated proteinase, calpain. Forced expression of calpain or exposure of cells to the calcium ionophore, A23187, increased CCTα degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCTα degradation. The effects of Ox-LDL on CCTα breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCTα isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCTα primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCTα generated p26, which upon NH2-terminal sequencing localized a calpain attack site within the CCTα amino terminus. Expression of a CCTα mutant where the amino-terminal cleavage site and a putative carboxyl-terminal hydrolysis region were modified resulted in an enzyme that was significantly less sensitive to proteolytic cleavage and restored the ability of cells to synthesize surfactant PtdCho after Ox-LDL treatment. Thus, these results provide a critical link between proatherogenic lipoproteins and their metabolic target, CCTα, resulting in impaired surfactant metabolism. Phosphatidylcholine (PtdCho) 1The abbreviations used are: PtdCho, phosphatidylcholine; CCT, CTP:phosphocholine cytidylyltransferase; LDL, low density lipoprotein; Ox-LDL, oxidized low density lipoproteins; VLDL, very low density lipoprotein; Ox-VLDL, oxidized very low density lipoproteins; LPDS, lipoprotein-deficient serum; MLE-12, murine lung epithelia; CHO, Chinese hamster ovary; CDP-choline, cytidine diphosphocholine; DSPtd-Cho, disaturated phosphatidylcholine; ALLN, N-acetyl-Leu-Leu-Nle-CHO; LysoPtdCho, lysophosphatidylcholine; ANOVA, analysis of variance; MALDI-MS, matrix-assisted laser desorption ion mass spectrometry; CMV, cytomegalovirus. has diverse biologic roles in mammalian cells and serves as the major phospholipid of pulmonary alveolar surfactant. Surfactant is synthesized and secreted from alveolar type II epithelial cells in a lipoproteinaceous form highly enriched with disaturated phosphatidylcholine (DSPtdCho), a critical surface-active component (1Rooney S.A. Am. Rev. Respir. Dis. 1985; 131: 439-460PubMed Google Scholar). DSPtdCho biosynthesis requires the uptake of choline into alveolar type II epithelial cells with the subsequent entry of this metabolite into the CDP-choline pathway (2Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar). A key step in this pathway involves the conversion of cholinephosphate to CDP-choline, which is catalyzed by the rate-limiting enzyme CTP: phosphocholine cytidylyltransferase (CCT) (EC 2.7.7.15 (2Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar)). CCT is an amphitropic enzyme, thus exhibiting interconversion between its soluble and membrane-associated forms. Accordingly, CCT localizes to several intracellular organelles, most notably the nuclear envelope and endoplasmic reticulum (3Wang Y. Sweitzer T.D. Weinhold P.A. Kent C. J. Biol. Chem. 1993; 268: 5899-5904Abstract Full Text PDF PubMed Google Scholar, 4Ridsdale R. Tseu I. Wang J. Post M. J. Biol. Chem. 2001; 276: 49148-49155Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). CCT activity is largely controlled by association with membrane lipids, and lipid regulation is well documented (2Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar). For example, anionic phospholipids, diacylglycerol, and unsaturated fatty acids all have been shown to potently activate the enzyme in vitro (5Yang W. Jackowski S. J. Biol. Chem. 1995; 270: 16503-16506Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 6Pelech S.L. Pritchard P.H. Brindley D.N. Vance D.E. J. Biol. Chem. 1983; 258: 6782-6788Abstract Full Text PDF PubMed Google Scholar, 7Feldman D.A. Kovac C.R. Dranginis P.L. Weinhold P.A. J. Biol. Chem. 1978; 253: 4980-4986Abstract Full Text PDF PubMed Google Scholar). CCT is also a phosphoenzyme, and membrane activation of CCT is influenced by enzyme phosphorylation status (5Yang W. Jackowski S. 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Three CCT isoforms exist in cells: CCTα, CCTβ1, and CCTβ2 (13Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). CCTα, the predominant species in alveolar epithelia contains four distinct functional domains including an amino-terminal nuclear localization domain, a mid-portion catalytic sequence, a lipid-binding domain, and a carboxyl-terminal phosphorylation domain. A second lipid-interacting domain within the carboxyl terminus of CCTα has been identified recently (14Lykidis A. Jackson P. Jackowski S. Biochemistry. 2001; 40: 494-503Crossref PubMed Scopus (42) Google Scholar). All CCT isoforms are catalytically active and are regulated by the availability of exogenous lipids. Studies in our laboratory have demonstrated that circulating native lipoproteins provide an important source of regulatory lipids for CCT activation and surfactant PtdCho synthesis (15Mallampalli R.K. Salome R.G. Bowen S.L. Chappell D.A. J. Clin. Invest. 1997; 99: 2020-2029Crossref PubMed Scopus (23) Google Scholar, 16Ryan A.J. Medh J D. McCoy D.M. Salome R.G. Mallampalli R.K. Am. J. Physiol. 2002; 283: L310-L318Crossref PubMed Scopus (22) Google Scholar). Very low density lipoproteins (VLDL) stimulate CCT activity and DSPtdCho synthesis in vivo (16Ryan A.J. Medh J D. McCoy D.M. Salome R.G. Mallampalli R.K. Am. J. Physiol. 2002; 283: L310-L318Crossref PubMed Scopus (22) Google Scholar). However, all lipoproteins deteriorate through an oxidative process, and lipoprotein oxidation is of central importance in the pathogenesis of atherosclerotic heart disease (17Jialal I. Devaraj S. Clin. Chem. 1996; 42: 498-506Crossref PubMed Scopus (205) Google Scholar). Low density lipoproteins (LDL) oxidize in the arterial intima in the presence of transition metals, thereby stimulating leukocyte-endothelial adhesion and recruitment of macrophages via chemoattractants. Severely oxidized LDL (Ox-LDL) is no longer recognized by the classic LDL receptor pathway, which is subject to feedback inhibition by intracellular cholesterol content but is taken up by macrophages via scavenger receptors. This in turn leads to the generation of the foam cell and the evolution of the fatty streak, hallmarks of atherosclerosis. Oxidized lipoproteins also appear to participate in the pathogenesis of several lung disorders such as asthma, acute lung injury, and cystic fibrosis (18Schunemann H.J. Muti P. Freudenheim J.L. Armstrong D. Browne R. Klocke R.A. Trevisan M. Am. J. Epidemiol. 1997; 146: 939-948Crossref PubMed Scopus (106) Google Scholar). Many of these disorders are characterized by decreased levels of surfactant PtdCho (19Jobe A.H. Ikegami M. Proc. Assoc. Am. Physicians. 1998; 110: 489-495PubMed Google Scholar). For example, in acute lung injury there is leakage of native lipoproteins from serum into the alveolar space (20Emmett M. Fowler A.A. Hyers T.M. Crowle A.J. Proc. Soc. Exp. Biol. Med. 1987; 184: 83-91Crossref PubMed Scopus (10) Google Scholar). These native lipoproteins are modified by an oxidative stress, intrinsic to lung injury, resulting from impaired antioxidant defenses, the presence of reactive oxidant species, and exposure to hyperoxia during mechanical ventilation (20Emmett M. Fowler A.A. Hyers T.M. Crowle A.J. Proc. Soc. Exp. Biol. Med. 1987; 184: 83-91Crossref PubMed Scopus (10) Google Scholar). Thus, pulmonary oxidative modification of native lipoproteins may be a critical event leading to altered surfactant lipid composition. Interestingly, alveolar type II epithelial cells express scavenger receptors that mediate uptake of antioxidants (21Kolleck I. Schlame M. Fechner H. Looman A.C. Wissel H. Rustow B. Free Radic. Biol. Med. 1999; 27: 882-890Crossref PubMed Scopus (94) Google Scholar). Collectively, these observations led us to hypothesize that Ox-LDL catabolism by alveolar epithelia might down-regulate surfactant DSPtdCho biosynthesis. To test this hypothesis, we determined the effects of oxidized lipoproteins on a key regulatory step within the CDP-choline pathway. We observed that these modified lipoproteins inhibit PtdCho synthesis in alveolar epithelia by triggering CCTα degradation via calpain-mediated cleavage of the enzyme. Materials—VLDL and LDL were purchased from Intracel (Frederick, MD). Lipoprotein-deficient serum (LPDS) (d > 1.21 g/ml) was isolated by ultracentrifugation (22Chappell D.A. Fry G.L. Waknitz M.A. Muhonen L.E. Pladet M.W. J. Biol. Chem. 1993; 268: 25487-25493Abstract Full Text PDF PubMed Google Scholar). The MLE-12 and CHO cell lines were purchased from American Type Culture Collection (Manassas, VA). Radio-labeled lipoproteins were prepared as described (15Mallampalli R.K. Salome R.G. Bowen S.L. Chappell D.A. J. Clin. Invest. 1997; 99: 2020-2029Crossref PubMed Scopus (23) Google Scholar). Lactacystin, A23187, N-acetyl-Leu-Leu-Nle-CHO (ALLN), recombinant rat calpain II, and calpastatin were purchased from Calbiochem (La Jolla, CA). A rabbit polyclonal CCTα antibody to synthetic peptide (10Mallampalli R.K. Ryan A.J. Salome R.G. Jackowski S. J. Biol. Chem. 2000; 275: 9699-9708Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) was generated by Covance Research Products Inc. (Richmond, CA), and rabbit polyclonal antibodies to M- and μ-calpain were from ABR-Affinity BioReagents (Golden, CO). The X-blue cells were from Stratagene (La Jolla, CA). The Taqman reverse transcription reagents, SYBR Green PCR master mix, and ProBlot were from Applied Biosystems (Foster City, CA). The pCR-TOPO4 plasmids and Escherichia coli Top10 competent cells were obtained from Invitrogen (Carlsbad, CA), and FuGENE6 transfection reagent was purchased from Roche Diagnostics. The Geneclean2 Kit was obtained from Bio101 (Carlsbad, CA). Recombinant histidine-tagged CCTα was kindly provided by Dr. Suzy Jackowski (23Luche M.M. Rock C.O. Jackowski S. Arch. Biochem. Biophys. 1993; 301: 114-118Crossref PubMed Scopus (26) Google Scholar). The plasmids pCMV5-CCT236 and pCMV5-CCT314 were kindly provided by Dr. Claudia Kent (24Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The pOP13-JHCPIS (calpastatin-inhibitory domain fragment) plasmid was a gift from Dr. Neil Forsberg (25Huang J. Forsberg N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12100-12105Crossref PubMed Scopus (268) Google Scholar). Calpain-deficient (Capn –/–) and wild-type (Capn +/+) embryonic fibroblasts were kindly provided by Dr. Peter Greer (26Arthur J.S. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell. Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (296) Google Scholar). The QuikChange site-directed mutagenesis kit was from Stratagene. The Advantage cDNA polymerase and the SMART™ cDNA library construction kit were from Clontech, (Palo Alto, CA). All DNA sequencing was performed by the University of Iowa DNA core facility. M-PER mammalian protein extraction reagent and the B-PER 6XHis spin purification kits were obtained from Pierce. NH2-terminal sequencing was performed by the Protein Core Laboratory at Michigan State University (East Lansing, MI). Lipoprotein Oxidation—Lipoproteins were oxidized by dialysis in phosphate-buffered saline containing 5 μm CuSO4 for24hat37 °C (27Esterbauer H. Gebicki J. Puhl H. Jurgens G. Free Radic. Biol. Med. 1992; 13: 341-390Crossref PubMed Scopus (2150) Google Scholar). Confirmation of lipoprotein oxidization was by the malonaldehyde assay and by apoprotein B-100 immunoblotting. Cell Isolation and Culture—Primary rat type II epithelial cells were isolated as described (28Longo C.A. Tyler D. Mallampalli R.K. Am. J. Respir. Cell Mol. Biol. 1997; 16: 605-612Crossref PubMed Scopus (36) Google Scholar). Cells were cultured in Dulbecco's minimum essential medium containing 10% LPDS for up to 24 h. MLE cells were cultured in Hite's medium containing LPDS for up to 48 h with or without LDL (100 μg/ml), Ox-LDL (10–100 μg/ml), or Ox-VLDL (10–100 μg/ml) (29Balibrea-Cantero J.L. Arias-Diaz J. Garcia C. Torres-Melero J. Simon C. Rodriguez J.M. Vara E. Am. J. Respir. Crit. Care Med. 1994; 149: 699-706Crossref PubMed Scopus (39) Google Scholar). In some studies, cells were preincubated with lactacystin (5 μm) or ALLN (40 μg/ml) for 1 h prior to exposure of cells to Ox-LDL. Cells lysates were isolated after brief sonication in Buffer A (10Mallampalli R.K. Ryan A.J. Salome R.G. Jackowski S. J. Biol. Chem. 2000; 275: 9699-9708Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) at 4 °C prior to analysis. Cell Surface Binding, Internalization, and Degradation of Oxidized Lipoproteins—Cellular catabolism of lipoproteins was determined as described (15Mallampalli R.K. Salome R.G. Bowen S.L. Chappell D.A. J. Clin. Invest. 1997; 99: 2020-2029Crossref PubMed Scopus (23) Google Scholar). Surface binding and internalization were defined as radioactivity that was released or remained cell-associated, respectively, following incubation of cells at 4 °C in buffer containing 10 mg/ml tripolyphosphate. Degradation was defined as the trichloroacetic acid-soluble radioactivity in the medium. PtdCho and DSPtdCho Analysis—Cells were pulsed with 1 μCi of [methyl-3H]choline chloride during the final 2 h of incubation with or without lipoproteins. Total cellular lipids were extracted (30Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43088) Google Scholar) and spotted on LK5D plates, and PtdCho was resolved using thin layer chromatography (10Mallampalli R.K. Ryan A.J. Salome R.G. Jackowski S. J. Biol. Chem. 2000; 275: 9699-9708Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). DSPtdCho determination was as described (31Mallampalli R.K. Salome R.G. Spector A.A. Am. J. Physiol. 1994; 267: L641—L648Google Scholar). Enzyme Assays—CCT activity was determined by measuring the rate of incorporation of [methyl-14C] phosphocholine into CDP-choline using a charcoal extraction method (32Mallampalli R.K. Mathur S.N. Warnock L.J. Salome R.G. Hunninghake G.W. Field F.J. Biochem. J. 1996; 318: 333-341Crossref PubMed Scopus (29) Google Scholar). Immunoblot Analysis—Equal amounts of total protein from cell lysates, adjusted to give a final concentration of 60 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromphenol blue, and 5% β-mercaptoethanol, were heated at 100 °C for 5 min. Samples were then electrophoresed through a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoreactive CCTα and calpain were detected using the ECL Western blotting detection system. The dilution factors for all polyclonal antibodies were 1:1000. [35S]Methionine Pulse-Chase and CCTα Immunoprecipitation— Turnover of CCTα was determined by preincubating MLE cells for 1 h in methionine-deficient medium and then pulsing with [35S]methionine (60 μCi/ml) for 4 h at 37 °C (10Mallampalli R.K. Ryan A.J. Salome R.G. Jackowski S. J. Biol. Chem. 2000; 275: 9699-9708Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Cells were rinsed twice in a similar medium and chased with serum-free Hite's medium containing 10 mm methionine and 3 mm cysteine for 0–24 h, with or without Ox-LDL. Cells were harvested and CCTα immunoprecipitated using the CCTα polyclonal antibody prior to separation using SDS-PAGE (10Mallampalli R.K. Ryan A.J. Salome R.G. Jackowski S. J. Biol. Chem. 2000; 275: 9699-9708Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Cloning of Rat CCTα—Total cellular RNA was isolated from primary rat adult type II alveolar epithelial cells using Tri-Reagent per the manufacturer's instructions. Double-stranded cDNA was generated from RNA using the SMART™ cDNA library construction kit following the manufacturer's instructions. The cDNA encoding the open reading frame for CCTα was generated using double-stranded cDNA as a template and using the sense primer 5′-agatctatggatgcacagagttcag-3′ and antisense primer 5′-tctagattagtcctcttcatcctcgctg-3′ in a two-step PCR amplification using Advantage cDNA polymerase. The reaction conditions were as follows: 94 °C for 2 min; 94 °C for 30 s, 68 °C for 3 min, 18 cycles. The ∼1100-bp CCTα open reading frame was purified using the Geneclean2 kit and cloned into pCR4-TOPO, and plasmid minipreps were verified by sequencing. This clone was then digested by BgIII and XbaI, purified by Geneclean2, and ligated into a pCMV5 expression vector previously digested with the same restriction enzymes. Construction of CCTα Mutants—An internal deletion CCTα mutant (termed CCTα289), lacking the putative membrane-binding domain of CCTα (amino acid residues 240–290), was generated as follows. pCMV5-CCTα was used as a template for PCR using the sense primer 5′-ggagctcaatgtcagctttatcaacagtcccaagcacagtccc-3′ and antisense primer 5′-tctagattagtcctcttcatcctcgctg-3′ to generate a 230-bp fragment that was purified and directionally cloned into pCR4-TOPO for transformation into TOP10-competent cells. pCMV5-CCTα was digested by SacI and XbaI, whereas pCR4-TOPO-CCTα was digested by BglII and SacI, generating 234- and 687-bp fragments, respectively. These products were purified and ligated into the BglII/XbaI site in pCMV5 using T4 ligase at 15 °C overnight. An amino-terminal CCTα mutant (CCTαSM), in which Ser5-Ser6 were mutated to Met5-Met6, was generated using the QuikChange site-directed mutagenesis kit. The oligonucleotides used were 5′-ctatggatgc acagatgatg gctaaagtca attcaagg-3′ (sense) and 5′-ccttgaattg actttagcca tcatctgtgc atccatag-3′ (antisense), and pCMV5-CCT plasmid DNA was used as a template. PCR conditions were as follows: 95 °C for 30 s, 18 cycles at 95 °C for 30 s, 55 °C for 60 s, and 68 °C for 6 min. We also generated a CCTα protein, termed CCTαPenta, that harbored the amino-terminal mutation above but also had residues Lys238-Lys239-Tyr240 mutated to Arg238-Arg239-Phe240, using procedures similar to those described above. The oligonucleotides used were 5′-gct tta tca acg aaa gga gat tcc act tgc aag aac g-3′ (sense) and 5′-cgt tct tgc aag tgg aat ctc ctt tcg ttg ata aag c-3′ (antisense), with CCTαSM plasmid DNA used as a template. The CCTαPenta construct was verified by DNA sequencing. Construction of Histidine-tagged CCTα and CCTα 289 Expression Vectors—Carboxyl-terminal histidine-tagged full-length CCTα and the deletion mutant CCTα289 vectors were generated by PCR using pCMV5-CCTα or pCMV5-CCTα289 as templates with the sense primer 5′-agatctatggatgcacagagttcag-3′ and antisense primer 5-′tctagatcaatgatgatgatgatgatggtcctcttcatcctcgc-3′. The resulting ∼1100- and ∼900-bp PCR products first were cloned into pCR4-TOPO and then ligated into the BglII and XbaI sites of pCMV5. The pCMV5-CCTα-His and pCMV5-CCTα289-His were verified by DNA sequencing. Purification of Recombinant pCMV5-CCTα-His and CCTα 289-His Proteins—The pCMV5-CCTα-His and CCTα289-His plasmids were transiently transfected into CHO cells. After 24 h, cell lysates were harvested using the M-PER mammalian protein extraction reagent. CCTα-histidine tag proteins were purified using the B-PER 6XHis spin purification kit following the manufacturer's instructions. Construction of M-calpain by Site-directed Mutagenesis—Mutagenesis of the M-calpain expression vector (pOP13-JHMS (25Huang J. Forsberg N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12100-12105Crossref PubMed Scopus (268) Google Scholar)) was performed by using the QuikChange site-directed mutagenesis kit and the primers 5′-ggagcccttggggactgctggcttctggct-3′ and 5′-agccagaagccagcagtccccaagggctcc-3′. The PCR conditions were: 95 °C for 30 s, 18 cycles at 95 °C for 30 s, 55 °C for 60 s, and 68 °C for 9 min. The resulting pOP13-calpain was verified by DNA sequencing. Real-time PCR Analysis—Total cellular RNA from MLE cells was obtained using Tri-Reagent. Taqman reverse transcription reagents were used to generate cDNA from cellular RNA. Real-time PCR was then performed on cDNA using the Applied Biosystems 7700 real-time PCR instrument and the SYBR Green PCR master mix. M-calpain mRNA detection primers were: 5′ primer, 5′-caagtcatcgttgcccgg-3′, and 3′ primer, 5′-ccaaacaccgcacaaaattg-3′. The μ-calpain 5′ primer was 5′-tcacccgctactcggagc-3′, and 3′ primer was 5′-cgcacaagacagcacacaaa-3′. Taqman rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control reagents were used as the internal control. Standard curves generated for calpain and compared with GAPDH using serial dilutions of mRNA were found to be linear from 0.08 ng to 50 ng RNA in the reaction mixture. This range included effective concentrations used in experiments. Transient Transfection and Over-expression of Recombinant Proteins—Transfections were conducted for 120 min in 0% fetal bovine serum medium using 10 μl of FuGENE 6™ reagent and 5 μg/dish of the desired plasmid. Immediately after transfections, cells were transferred to medium containing 2–10% fetal bovine serum and allowed to recover for 24 h before cell lysates were harvested for analysis. In other studies, cells were transfected with pOP13-JHCPIS (calpastatin inhibitory domain fragment) with or without Ox-LDL (100 μg/ml) for 48 h or with pOP13-calpain. In Vitro Digestion of CCTα—Digestions were conducted at 35 °C for 20 min in 50-μl reaction mixtures containing 25 μg of purified rat CCTα-His and 0.7 μg of recombinant rat M-calpain in calpain buffer (20 mm Tris, pH 7.5, 2 mm dithiothreitol, 1% Tween 20, and 0.015% Triton X-100). The reaction was started by adding CaCl2 to a final concentration of 3 mm and was terminated by adding 10 mm EDTA and then heating the samples to 95 °C. After digested proteins were resolved by SDS-PAGE, they were transferred to Problot membranes for Coomassie Blue staining and submitted to NH2-terminal sequencing or processed for immunoblotting. Statistical Analysis—Statistical analysis was performed using one-way ANOVA with a Bonferroni adjustment or Student's unpaired t test (33Rosner B.A. Fundamentals of Biostatistics. Wadsworth Publishing Co., Belmont, CA1995: 314-318Google Scholar). Oxidized Lipoprotein Catabolism by Alveolar Epithelia— MLE cells bound, internalized, and degraded 125I-labeled oxidized human LDL and oxidized VLDL. Cells exhibited a nearly 8-, 4-, and 6-fold increase in steady-state uptake, internalization, and degradation of 125I-Ox-LDL ligand, respectively (Fig. 1). Ox-LDL catabolism increased steadily up to concentrations of ∼25 μg/ml ligand, thereafter reaching a plateau at higher doses indicative of saturable kinetics (Fig. 1). Cells also exhibited a dose-dependent increase in catabolism of Ox-VLDL; however, saturability was not always observed within the range of lipoproteins tested in these studies (Fig. 1, insets). In preliminary studies, primary rat alveolar type II epithelial cells also bound and internalized oxidized lipoproteins (data not shown). Thus, alveolar epithelia actively engage in modified lipoprotein catabolism. Ox-LDL Inhibits Surfactant PtdCho Biosynthesis—Ox-LDL significantly decreased the incorporation of [methyl-3H]choline into PtdCho and DSPtdCho in primary type II epithelia and MLE cells. In primary cells, Ox-LDL significantly decreased choline incorporation into PtdCho by ∼40% after 24 h of exposure (Fig. 2A). Similar effects of Ox-LDL were observed on choline incorporation into DSPtdCho, the primary surface-active component of surfactant, which was reduced by 46% versus control (Fig 2A, inset). In MLE cells, the effects of Ox-LDL were more pronounced, as Ox-LDL inhibited choline incorporation into PtdCho and DSPtdCho by 75 and 67%, respectively (Fig. 2B and inset). To further investigate the effects of Ox-LDL on DSPtdCho synthesis, we assayed enzymes within the CDP-choline pathway. Ox-LDL produced a 43% decrease in CCT activity in primary cells (Fig. 2C) and a dose-dependent decrease in CCT activity in MLE cells (Fig. 2C, inset; n = 3, p < 0.05 versus control). In contrast, these particles tended to increase cholinephosphosphotransferase activity but did not alter choline kinase activity in primary cells (data not shown). Thus, Ox-LDL substantially reduces surfactant lipid biosynthesis by selectively inhibiting the rate-regulatory step within the CDP-choline pathway. Ox-LDL Increases CCTα Protein Turnover—Ox-LDL produced a dose-dependent decrease in the steady-state levels of the 42-kDa native CCTα protein after 48 h of exposure without having any effect on β-actin (Fig. 3, A and B). Similar effects on CCTα levels were seen after Ox-VLDL treatment of MLE cells (Fig. 3C) and in primary alveolar cells (Fig. 3D). These data suggest that Ox-LDL decreases PtdCho synthesis by increasing CCTα protein degradation. Confirmation of Ox-LDL effects on enzyme turnover was made using [35S]methionine pulse-chase studies. The amount of [35S]methionine incorporated into immunoprecipitable CCTα was determined after a 4-h pulse followed by a 0–24-h chase with unlabeled methionine conducted in the presence or absence of Ox-LDL. The amount of [35S]methionine incorporated into immunoprecipitable CCTα was decreased at 6 h by Ox-LDL (Fig. 3E), and by 18–24 h substantially less CCTα was observed in Ox-LDL-treated cells compared with control. Effects of Ox-LDL on CCTα Expression Are Mediated by Calpain—Cells were preincubated with ALLN and lactacystin, calpain, and 20 S proteasome inhibitors, respectively, and samples were analyzed for CCTα protein content and activity (Fig. 4, A and B). Pretreatment with either ALLN or lactacystin partly blocked Ox-LDL-induced CCTα degradation (Fig. 4A). The effects of ALLN were generally greater than lactacystin in antagonizing Ox-LDL on CCTα protein levels, although both inhibitors were equally effective in attenuating the lipoprotein-induced inhibition of CCT activity (Fig. 4, A and B). To investigate calcium-activated proteinases on CCTα degradation, cells were exposed to A23187, which stimulates calpain activity. Similar to Ox-LDL, the ionophore A23187 induced CCTα degradation (Fig. 4C). Calpain-mediated proteolysis in cells is tightly regulated by the availability of its endogenous inhibitor, calpastatin. When MLE cells were transfected with a plasmid encoding a calpastatin inhibitory domain fragment, the effects of Ox-LDL on CCTα degradation were reduced compared with cells transfected with a control plasmid (Fig. 4D). Further, overexpression of calpain in MLE cells induced CCTα degradation similar to calcium ionophore (Fig.
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