Biomedical subjects

Rama K Mallampalli

Publications and source records attributed to Rama K Mallampalli.

At least 19 recordsLinked to original sources

PubMed2007

Pseudomonas aeruginosa and sPLA2 IB stimulate ABCA1-mediated phospholipid efflux via ERK-activation of PPARalpha-RXR.

Bacterial infection triggers an acute inflammatory response that might alter phospholipid metabolism. We have investigated the acute-phase response of murine lung epithelia to Pseudomonas aeruginosa infection. Ps. aeruginosa triggered secretion of the pro-inflammatory lipase, sPLA2 IB (phospholipase A2 IB), from lung epithelium. Ps. aeruginosa and sPLA2 IB each stimulated basolateral PtdCho (phosphatidylcholine) efflux in lung epithelial cells. Pre-treatment of cells with glyburide, an inhibitor of the lipid-export pump, ABCA1 (ATP-binding cassette transporter A1), attenuated Ps. aeruginosa and sPLA2 IB stimulation of PtdCho efflux. Effects of Ps. aeruginosa and sPLA2 IB were completely abolished in human Tangier disease fibroblasts, cells that harbour an ABCA1 genetic defect. Ps. aeruginosa and sPLA2 IB induced the heterodimeric receptors, PPARa (peroxisome-proliferator-activated receptor-a) and RXR (retinoid X receptor), factors known to modulate ABCA1 gene expression. Ps. aeruginosa and sPLA2 IB stimulation of PtdCho efflux was blocked with PD98059, a p44/42 kinase inhibitor. Transfection with MEK1 (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase 1), a kinase upstream of p44/42, increased PPARa and RXR expression co-ordinately with increased ABCA1 protein. These results suggest that pro-inflammatory effects of Ps. aeruginosa involve release of an sPLA2 of epithelial origin that, in part, via distinct signalling molecules, transactivates the ABCA1 gene, leading to export of phospholipid.

ATP Binding Cassette Transporter 1↗

PubMed2006

Chronic Pseudomonas aeruginosa infection reduces surfactant levels by inhibiting its biosynthesis.

Chronic Pseudomonas aeruginosa infection, as occurs in cystic fibrosis, is associated with decreased surfactant phospholipid levels. To investigate mechanisms, we measured synthesis of dipalmitoylphosphatidylcholine (DPPC), the major surfactant phospholipid. Mice received an agarose bead slurry alone, or were infected with beads containing a clinical mucoid isolate of P. aeruginosa. Bacterial infection after 3 days resulted in a approximately 50% reduction in surfactant DPPC content versus control. These changes in surfactant were associated with co-ordinate reductions in mRNAs and immunoreactive levels for CTP: phosphocholine cytidylyltransferase (CCTalpha), the rate-regulatory enzyme required for DPPC synthesis. P. aeruginosa infection of murine lung epithelia decreased CCTalpha gene transcription without altering mRNA stability and by a mechanism other than release of a soluble extracellular inhibitor. Promoter deletional analysis revealed that P. aeruginosa activates a negative response element from -1019 to -799 bp of the CCTalpha proximal 5'-flanking region. Exposure of cells to a P. aeruginosa mutant strain producing alginate reduced CCTalpha promoter activity, whereas these effects were not observed in strains defective in alginate synthesis. Murine type II cells isolated from P. aeruginosa-infected CCTalpha promoter-beta-galactosidase transgenic mice exhibited significantly reduced CCT and beta-galactosidase enzyme activities versus control. Thus, a mucoid P. aeruginosa strain reduces mRNA synthesis of a key biosynthetic enzyme thereby decreasing levels of surfactant.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2006

Proapoptotic effects of P. aeruginosa involve inhibition of surfactant phosphatidylcholine synthesis.

Pseudomonas aeruginosa causes sepsis-induced acute lung injury, a disorder associated with deficiency of surfactant phosphatidylcholine (PtdCho). P. aeruginosa (PA103) utilizes a type III secretion system (TTSS) to induce programmed cell death. Herein, we observed that PA103 reduced alveolar PtdCho levels, resulting in impaired lung biophysical activity, an effect partly attributed to caspase-dependent cleavage of the key PtdCho biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha). Expression of recombinant CCTalpha variants harboring point mutations at putative caspase cleavage sites in murine lung epithelia resulted in partial proteolytic resistance of CCTalpha to PA103. Further, caspase-directed CCTalpha degradation, decreased PtdCho levels, and cell death in murine lung epithelia were lessened after exposure of cells to bacterial strains lacking the TTSS gene product, exotoxin U (ExoU), but not ExoT. These observations suggest that during the proapoptotic program driven by P. aeruginosa, deleterious effects on phospholipid metabolism are mediated by a TTSS in concert with caspase activation, resulting in proteolysis of a key surfactant biosynthetic enzyme.

Animals↗

PubMed2006

Transcriptional regulation of lung cytidylyltransferase in developing transgenic mice.

Lung development is associated with a surge in surfactant phosphatidylcholine (PC) production to prepare the newborn for extrauterine breathing. This process is associated with a marked increase in the activity of the rate-regulatory surfactant enzyme, CTP:phosphocholine cytidylyltransferase (CCTalpha). To investigate the molecular basis for developmental activation of CCTalpha, we analyzed expression of endogenous CCTalpha and a reporter gene, beta-galactosidase, in fetal, newborn, and adult promoter-reporter transgenic mice. Transgenics harboring approximately 2 kb of the CCTalpha promoter linked upstream of a beta-galactosidase reporter gene displayed relatively high expression in distal lung epithelia. Endogenous lung CCTalpha and beta-galactosidase activities, protein content, and transcript levels displayed maximal expression within the newborn period. CCTalpha and beta-galactosidase activities and enzyme levels increased with time in cultured fetal lung explants isolated from transgenics. Transfectional analysis using CCTalpha promoter-reporter constructs in developing rat type II cells revealed that a region encompassing -169/+71 contained the DNA elements required for perinatal activation. The studies demonstrate that developmental induction of surfactant phospholipid is due, at least in part, to transcriptional activation of the CCTalpha gene.

Animals↗

PubMed2006

c-Jun N-terminal kinase regulates CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CCTalpha) is a rate-regulatory enzyme required for phosphatidylcholine (PtdCho) synthesis. CCTalpha is also a phosphoenzyme, but the physiologic role of kinases on enzyme function remains unclear. We report high-level expression of two major isoforms of the c-Jun N-terminal kinase family (JNK1 and JNK2) in murine lung epithelia. Further, JNK1 and JNK2 phosphorylated purified CCTalpha in vitro, and this was associated with a dose-dependent decrease (approximately 40%) in CCT activity. To evaluate JNK in vivo, lung epithelial cells were infected with a replication defective adenoviral vector encoding murine JNK2 (Adv-JNK2) or an empty vector. Adv-JNK2 infection, unlike the empty vector, markedly increased JNK2 expression concomitant with increased incorporation of [32P]orthophosphate into endogenous CCTalpha. Although Adv-JNK2 infection only modestly reduced CCT activity, it reduced PtdCho synthesis by approximately 30% in cells. These observations suggest a role for JNK kinases as negative regulators of phospholipid synthesis in murine lung epithelia.

Animals↗

PubMed2005

Oxysterols inhibit phosphatidylcholine synthesis via ERK docking and phosphorylation of CTP:phosphocholine cytidylyltransferase.

Surfactant deficiency contributes to acute lung injury and may result from the elaboration of bioactive lipids such as oxysterols. We observed that the oxysterol 22-hydroxycholesterol (22-HC) in combination with its obligate partner, 9-cis-retinoic acid (9-cis-RA), decreased surfactant phosphatidylcholine (PtdCho) synthesis by increasing phosphorylation of the regulatory enzyme CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha). Phosphorylation of CCTalpha decreased its activity. 22-HC/9-cis-RA inhibition of PtdCho synthesis was blocked by PD98059 or dominant-negative ERK (p42 kinase). Overexpression of constitutively active MEK1, the kinase upstream of p42 kinase, increased CCTalpha phosphorylation. Expression of truncated CCTalpha mutants lacking proline-directed sites within the C-terminal phosphorylation domain partially blocked oxysterol-mediated inhibition of PtdCho synthesis. Mutagenesis of Ser315 within CCTalpha was both required and sufficient to confer significant resistance to 22-HC/9-cis-RA inhibition of PtdCho synthesis. A novel putative ERK-docking domain N-terminal to this phosphoacceptor site was mapped within the CCTalpha membrane-binding domain (residues 287-300). The results are the first demonstration of a physiologically relevant phosphorylation site and docking domain within CCTalpha that serve as targets for ERKs, resulting in inhibition of surfactant synthesis.

Alitretinoin↗

PubMed2005

LASS5 is the predominant ceramide synthase isoform involved in de novo sphingolipid synthesis in lung epithelia.

Ceramide is a key bioactive mediator that inhibits surfactant phosphatidylcholine (PtdCho) synthesis in lung epithelia. Ceramide availability is governed by sphingomyelin (SM) hydrolysis, but less is known regarding its de novo synthesis. In this study, we observed that ceramide synthesis within murine lung epithelia was associated with high-level ceramide synthase (dihydroceramide synthase) activity. Longevity assurance homolog 5 (LASS5) was the predominant ceramide synthase isoform detected in lung epithelia, whereas relatively lower level expression was detected for the other five mammalian homologs. Pulmonary LASS5 was developmentally regulated, but its expression was spatially and gender nonspecific. Exogenously expressed LASS5 in lung epithelia was membrane-associated, triggering increased ceramide synthesis, whereas knockdown studies using fumonisin B1 or LASS5 small, interfering RNA reduced ceramide synthase activity by 78% or 45%, respectively. Overexpression of LASS5 also reduced PtdCho synthesis, but maximal inhibition was achieved when LASS5 was coexpressed with a plasmid encoding a neutral sphingomyelinase involved in SM hydrolysis. These results demonstrate that LASS5 is the major ceramide synthase gene product involved in sphingolipid production that may also regulate PtdCho metabolism in pulmonary epithelia.

Animals↗

PubMed2005

Gene transfer to respiratory epithelia with lentivirus pseudotyped with Jaagsiekte sheep retrovirus envelope glycoprotein.

A feline immunodeficiency virus (FIV)-based lentiviral vector was pseudotyped to identify envelope (env) glycoproteins that direct efficient gene transfer to pulmonary epithelia for the treatment or prevention of lung diseases. The envelope glycoprotein from the Jaagsiekte sheep retrovirus (JSRV) is a candidate under investigation. We utilized high titer FIV vector (>10(8) TU/ml) pseudotyped with the JSRV env glycoprotein (JSRVFIV) to study the transduction of polarized primary cultures of human airway epithelia and receptor/vector interactions. The reported receptor for JSRV, hyaluronidase 2 (HYAL2), is a GPI-linked protein. We expressed FLAG-tagged HYAL2 in polarized airway epithelia using an adenoviral vector and documented that the HYAL2 protein sorts predominantly to the apical surface. Of interest, the efficiency of gene transfer with apically applied JSRV-FIV was markedly less than FIV pseudotyped with VSV-G, even in Ad-HYAL2 complemented epithelia. The inefficient gene transfer with JSRV-FIV in HYAL2 complemented cells suggests that factors other than receptor abundance limit apical gene transfer efficiency with this envelope. JSRV-FIV transduced the distal lung epithelia of rabbits in vivo and transduced primary cultures of rabbit type II cells with 100-fold greater efficiency than primary cultures of rabbit tracheal cells. These data indicate that a lentivirus pseudotyped with the JSRV envelope glycoprotein transduces type II cells with greater efficiency than conducting airway epithelia and provides an example of glycoprotein-mediated cell-specific tropism within a tissue with a widely heterogeneous cell population.

Animals↗

PubMed2004

Adenovirus stimulates choline efflux by increasing expression of organic cation transporter-2.

We examined the effect of wild-type human adenovirus (Ad5) on choline transport in murine lung epithelia (MLE) and in rodent primary alveolar type II cells. Cells were active in pH-sensitive, reversible transport of choline, a process blocked pharmacologically with phenoxybenzamine, an inhibitor of organic cation transporters (OCT). PCR products for the choline transporters, OCT-1 and OCT-2, were detected, but only OCT-2 protein was robustly expressed within MLE and primary alveolar epithelial cells. Ad5 produced a two- to threefold increase in choline efflux from cells, resulting in a significant reduction in intracellular choline content and its major product, phosphatidylcholine. Effects of Ad5 on choline efflux were inhibited with phenoxybenzamine, and choline efflux was attenuated by OCT-2 small interfering RNA. Adenovirus also produced a dose-dependent increase in immunoreactive OCT-2 levels concomitant with increased cellular OCT-2 steady-state mRNA. These results indicate that adenoviruses can significantly disrupt choline trafficking in lung epithelia by upregulating expression of an alveolar protein involved in organic cation transport.

Adenoviridae↗

PubMed2004

Transcriptional repression of the CTP:phosphocholine cytidylyltransferase gene by sphingosine.

We examined the effects of the bioactive lipid, sphingosine, on the expression of the rate-limiting enzyme involved in surfactant phosphatidylcholine synthesis, CCTalpha (CTP:phosphocholine cytidylyltransferase alpha). Sphingosine decreased phosphatidylcholine synthesis by inhibiting CCT activity in primary alveolar type II epithelia. Sphingosine decreased CCTalpha protein and mRNA levels by approx. 50% compared with control. The bioactive lipid did not alter CCTalpha mRNA stability, but significantly inhibited its transcriptional rate. In murine lung epithelia, sphingosine selectively reduced CCTalpha promoter-reporter activity when transfected with a 2 kb CCTalpha promoter/luciferase gene construct. Sphingosine also decreased transgene expression in murine type II epithelia isolated from CCTalpha promoter-reporter transgenic mice harbouring this 2 kb proximal 5'-flanking sequence. Deletional analysis revealed that sphingosine responsiveness was mapped to a negative regulatory element contained within 814 bp upstream of the coding region. The results indicate that bioactive sphingolipid metabolites suppress surfactant lipid synthesis by inhibiting gene transcription of a key surfactant biosynthetic enzyme.

Animals↗

PubMed2004

Cooperative prosurvival activity by ERK and Akt in human alveolar macrophages is dependent on high levels of acid ceramidase activity.

Human alveolar macrophages are unique in that they have an extended life span in contrast to precursor monocytes. In evaluating the role of sphingolipids in alveolar macrophage survival, we found high levels of sphingosine, but not sphingosine-1-phosphate. Sphingosine is generated by the action of ceramidase(s) on ceramide, and alveolar macrophages have high constitutive levels of acid ceramidase mRNA, protein, and activity. The high levels of acid ceramidase were specific to alveolar macrophages, because there was little ceramidase protein or activity (or sphingosine) in monocytes from matching donors. In evaluating prolonged survival of alveolar macrophages, we observed a requirement for constitutive activity of ERK MAPK and the PI3K downstream effector Akt. Blocking acid ceramidase but not sphingosine kinase activity in alveolar macrophages led to decreased ERK and Akt activity and induction of cell death. These studies suggest an important role for sphingolipids in prolonging survival of human alveolar macrophages via distinct survival pathways.

Cell Survival↗

PubMed2004

Upregulation of surfactant synthesis triggers ABCA1-mediated basolateral phospholipid efflux.

Alveolar type II lung epithelia produce surfactant, an essential surface-active material highly enriched with disaturated phosphatidylcholine (DSPC), which requires a key regulatory enzyme, CTP:phosphocholine cytidylyltransferase alpha (CCTalpha), for its synthesis before its export apically into the alveolus. In this study, we examined whether surfactant phosphatidylcholine (PC) synthesis and export are physiologically linked. Stable overexpression of CCTalpha in lung epithelial cell lines increased rates of PC synthesis and cellular DSPC mass without altering total cellular PC content. Overexpression of CCTalpha was associated with i) increased basolateral, rather than apical, PC export catalyzed by ABCA1; ii) basolateral export of significant levels of unsaturated (nonsurfactant) PC; and iii) transcriptional activation of the ABCA1 gene via a liver X receptor/retinoic acid receptor-independent pathway. Cells exposed to PC vesicles exhibited a dose-dependent increase in ABCA1 transcriptional activity. These data provide the first evidence that surfactant PC synthesis is linked to its export via a basolateral lipid efflux pathway. This pathway is mediated, in part, by a phospholipid sensor, ABCA1, that appears to partake in the autoregulation of both cellular content and composition of PC, thereby providing a potentially novel exit route for a newly synthesized pool of PC distinct from surfactant.

ATP Binding Cassette Transporter 1↗

PubMed2004

Oxysterols trigger ABCA1-mediated basolateral surfactant efflux.

Surfactant is an apically-secreted surface-active material containing primarily disaturated phosphatidylcholine (DSPtdCho) that is released from alveolar epithelia into the alveolus. Surfactant deficiency is an important aspect of inflammatory lung disease and may result from extravasation of serum lipoproteins into the alveolus. We investigated whether one bioactive component of modified lipoproteins, oxysterols, might reduce surfactant PtdCho availability by altering its trafficking. The oxysterol, 22-hydroxycholesterol (22HC), in combination with its obligate partner, 9 cis-retinoic acid (RA), decreased surfactant PtdCho levels, in part, by stimulating basolateral phospholipid export in murine lung epithelia. 22HC/RA stimulated basolateral PtdCho efflux in cells via transcriptional activation of the ATP-binding cassette transporter 1 (ABCA1) gene. This effect was mediated by a DR-4 locus within the ABCA1 promoter. ABCA1 knockdown studies using ABCA1 siRNA or the ABCA1 inhibitor, glyburide, selectively attenuated 22HC/RA-driven basolateral PtdCho efflux. 22HC/RA significantly increased export of PtdCho molecular species containing saturated (16:0) fatty-acyl species typical of DSPtdCho. Overexpression of ABCA1 mimicked 22HC/RA effects by increasing cellular PtdCho efflux, whereas mutagenesis of ABCA1 at Trp590 attenuated PtdCho release. The results indicate the existence of an oxysterol-activated basolateral exit pathway for surfactant that might impact the availability of phospholipid destined for apical secretion.

ATP Binding Cassette Transporter 1↗

PubMed2004

Sphingosine kinase mediates activation of extracellular signal-related kinase and Akt by respiratory syncytial virus.

Respiratory syncytial virus (RSV) preferentially infects lung epithelial cells. Infected cells remain viable well into the infection. This prolonged survival results from RSV-induced activation of pro-survival pathways, including Akt and extracellular signal-related kinase (ERK). Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite with demonstrated links to cell survival. It is enzymatically generated by sequential activation of ceramidase (generation of sphingosine) and sphingosine kinase (generation of S1P). In these studies, we found that RSV stimulated neutral ceramidase and sphingosine kinase activities in lung epithelial cells. The combined effect of activation of these two enzymes would decrease proapoptotic ceramide and increase antiapoptotic S1P. S1P activated Akt and ERK within minutes, and inhibition of sphingosine kinase blocked RSV-induced ERK and Akt activation, leading to accelerated cell death after viral infection. RSV infection does eventually kill infected cells but activation of cell survival pathways significantly delays cell death. The studies are the first evidence linking sphingolipid metabolites to cell survival mechanisms in the context of a viral infection.

Amidohydrolases↗

PubMed2003

Oxidized lipoproteins inhibit surfactant phosphatidylcholine synthesis via calpain-mediated cleavage of CTP:phosphocholine cytidylyltransferase.

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 CCTalpha 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 CCTalpha degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCTalpha degradation. The effects of Ox-LDL on CCTalpha breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCTalpha isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCTalpha primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCTalpha generated p26, which upon NH2-terminal sequencing localized a calpain attack site within the CCTalpha amino terminus. Expression of a CCTalpha 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, CCTalpha, resulting in impaired surfactant metabolism.

Amino Acid Sequence↗

PubMed2003

The CCT promoter directs high-level transgene expression in distal lung epithelial cell lines.

Gene therapy requires the presence of a robust and yet small promoter to drive high-level expression of desired proteins. In comparative analysis, we investigated the promoter strength of the CTP:phosphocholine cytidylyltransferase promoter (CCT alpha) with other commonly used promoters, which were all cloned into a similar background vector (PGL3 basic). Transient promoter-reporter assays in murine lung epithelial (MLE-12) cells revealed that the core CCT alpha promoter (240 bp) was observed to exhibit a 40-fold, 8-fold, and 3-fold higher level of activity compared with the simian virus 40, human cytomegalovirus, and Rous sarcoma virus promoters, respectively. The CCT alpha promoter was significantly more active than the Clara cell 10, thymidine kinase, and phosphoglycerate kinase promoters. This pattern of high-level expression for CCT alpha was detected primarily in cell lines of distal lung epithelial origin (MLE-12, RLE, H441) and was reduced in other cell lines (A549, CHO, HepG 2). CCT alpha promoter-reporter activity, CCT alpha transcript levels, and immunoreactive protein levels increased significantly in the presence of all-trans retinoic acid. The CCT alpha promoter, in a retinoic acid-inducible manner, efficiently directed expression of murine erythropoietin in MLE-12 cells. Collectively, these observations suggest that the CCT alpha construct might be useful to drive high-level, regulatable expression of heterologous proteins in alveolar epithelia.

Animals↗

PubMed2002

Alveolar sphingolipids generated in response to TNF-alpha modifies surfactant biophysical activity.

Sphingolipids represent a diverse group of bioactive lipid species that are generated intracellularly in response to tumor necrosis factor-alpha (TNF-alpha) and are implicated as potential mediators of acute lung injury. The purpose of these studies was to determine whether there was an extracellular, TNF-alpha-regulated pool of sphingolipids in the alveolus that modulates the surface tension lowering capacity of pulmonary surfactant. Intratracheal instillation of TNF-alpha in adult rats led to a twofold increase in the amount of surfactant-associated ceramide and tended to decrease levels of sphingomyelin without significantly altering sphingosine or sphinganine content. TNF-alpha induction of alveolar ceramide was associated with nearly an 80% increase in acid sphingomyelinase activity recovered in cell-free alveolar lavage. Ceramide administered in a dose-dependent manner potently antagonized the surface tension lowering effects of natural surfactant in vitro. Intratracheal TNF-alpha and ceramide treatment of rats significantly increased lung permeability, as was evidenced by extravasation of Evans blue dye into alveolar lavage and lung tissue. Thus these studies are the first to demonstrate the existence of a cytokine-regulated alveolar pool of sphingomyelin hydrolysis products that impairs the biophysical properties of the alveolar surfactant film. The results also suggest the presence of a secretory alveolar sphingomylinase that is TNF-alpha responsive and mediates effects of the cytokine on alveolar sphingolipid metabolism.

Animals↗

PubMed2002

Lipid deprivation increases surfactant phosphatidylcholine synthesis via a sterol-sensitive regulatory element within the CTP:phosphocholine cytidylyltransferase promoter.

Lipid-deprived mice increase alveolar surfactant disaturated phosphatidylcholine (DSPtdCho) synthesis compared with mice fed a standard diet by increasing expression of CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme for DSPtdCho synthesis. We previously observed that lipid deprivation increases mRNA synthesis for CCT [Ryan, McCoy, Mathur, Field and Mallampalli (2000) J. Lipid Res. 41, 1268-1277]. To evaluate regulatory mechanisms for this gene, we cloned the proximal approximately 1900 bp of the 5' flanking sequence of the murine CCT gene, coupled this to a luciferase reporter, and examined transcriptional regulation in a murine alveolar epithelial type II cell line (MLE-12). The core promoter was localized to a region between -169 and +71 bp, which exhibited strong basal activity comparable with the simian virus 40 promoter. The full-length construct, from -1867 to +71, was induced 2-3-fold when cells were cultured in lipoprotein-deficient serum (LPDS), similar to the level of induction of the endogenous CCT gene. By deletional analysis the sterol regulatory element (SRE) was localized within a 240 bp region. LPDS activation of the CCT promoter was abolished by mutation of this SRE, and gel mobility-shift assays demonstrated specific binding of recombinant SRE-binding protein to this element within the CCT promoter. These observations indicate that sterol-regulated expression of CCT is mediated by an SRE within its 5' flanking region.

Animals↗