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MUCOSAL BIOLOGY

Modulation of human Niemann-Pick C1-like 1 gene expression by sterol: role of sterol regulatory element binding protein 2

Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois, and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois

Submitted 11 July 2006 ; accepted in final form 24 September 2006

	ABSTRACT

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Niemann-Pick C1-like 1 (NPC1L1) is an essential intestinal component of cholesterol absorption. However, little is known about the molecular regulation of intestinal NPC1L1 expression and promoter activity. We demonstrated that human NPC1L1 mRNA expression was significantly decreased by 25-hydroxycholesterol but increased in response to cellular cholesterol depletion achieved by incubation with Mevinolin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase) in human intestinal Caco-2 cells. We also showed that a –1741/+56 fragment of the NPC1L1 gene demonstrated high promoter activity in Caco-2 cells that was reduced by 25-hydroxycholesterol and stimulated by cholesterol depletion. Interestingly, we showed that the NPC1L1 promoter is remarkably transactivated by the overexpression of sterol regulatory element (SRE) binding protein (SREBP)-2, suggesting its involvement in the sterol-induced alteration in NPC1L1 promoter activity. Finally, we identified two putative SREs in the human NPC1L1 promoter and established their essential roles in mediating the effects of cholesterol on promoter activity. Our study demonstrated the modulation of human NPC1L1 expression and promoter activity by cholesterol in a SREBP-2-dependent mechanism.

oxysterols; intestinal cholesterol absorption

Intestinal cholesterol absorption has been shown to be a major determinant of plasma levels of cholesterol (15). Also, ezetimibe, a selective inhibitor of intestinal cholesterol uptake, has been effectively used in the treatment of hypercholesterolemia (16). Although intestinal cholesterol absorption appears to be a multistep process, a recent study (9) has identified Niemann-Pick C1-like 1 (NPC1L1) protein as a direct molecular target for ezetimibe, indicating its pivotal role in the intestinal uptake of cholesterol. NPC1L1 protein is predominantly expressed in the liver and proximal intestine (2, 4) and shares 50% amino acid homology with Niemann-Pick C1, which is involved in intracellular cholesterol trafficking and is defective in Niemann-Pick type C cholesterol storage disease (3). Furthermore, NPC1L1 knockout mice exhibited an 70% reduction in cholesterol absorption and were insensitive to the effect of ezetimibe (2). A recent study (24) has successfully shown reconstitution of NPC1L1-dependent cholesterol transport in a cell culture system, lending further proof for its direct involvement in cholesterol uptake. Since NPC1L1 is an integral part of complex processes of cholesterol homeostasis, it is speculated that it is tightly regulated and that the regulation of its expression is harmonized with the modulation of other genes involved in cholesterol metabolism (6, 22).

The coordinated regulation of genes implicated in cholesterol homeostasis is governed by the actions of several transcription factors, such as liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR)-, which are influenced by plasma levels of cholesterol and other lipids. Also, sterol regulatory element (SRE) binding proteins (SREBPs) are transcription factors that are crucial regulators of cholesterol synthesis and metabolism. The roles of these transcription factors in the modulation of genes critical for the processes of cholesterol synthesis and its conversion to bile acids are well defined. However, their involvement in the modulation of intestinal cholesterol absorption and the expression of NPC1L1 protein are still not fully understood. Recently, the expression of murine NPC1L1 was shown to be significantly decreased in response to feeding of a cholesterol-enriched diet (5). Also, a study by Duval et al. (6) demonstrated the downregulation of human (h)NPC1L1 and murine NPC1L1 expression by LXR agonists. In these studies, however, the expression of NPC1L1 by a PPAR- agonist remained unaltered. On the other hand, a study by van der Veen et al. (22) has shown an inhibition in NPC1L1 expression in mice by PPAR- agonists. Collectively, these previous observations have indicated the modulation of NPC1L1 expression by cholesterol and the involvement of several nuclear receptors in this regulation. However, the exact molecular mechanism(s) by which cholesterol directly influences the expression of intestinal NPC1L1 is still unclear.

Therefore, the present study was undertaken to investigate the effect of cholesterol on the expression and promoter activity of intestinal hNPC1L1. Our results demonstrated that sterol addition to human intestinal Caco-2 cells led to a decrease, whereas cellular cholesterol depletion caused an increase, in the expression of NPC1L1 mRNA and its promoter activity. Also, our data showed the involvement of SREBP-2 in the observed regulation of intestinal hNPC1L1 by cholesterol. These findings provide novel evidence for the possible involvement of SREBP-2 in the regulation of intestinal cholesterol absorption.

	METHODS

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Cell culture. Caco-2 and human embryonic kidney (HEK)-293 cells were obtained from the American Type Culture Collection (ATCC) and were grown routinely in T-75-cm² plastic flasks at 37°C in a 5% CO₂-95% air environment. Cells were cultured in minimum essential medium (Eagle) with 2 mM L-glutamine and Earle’s balanced salt solution adjusted to contain 1.5 g/l sodium bicarbonate, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate and supplemented with FBS (20% for Caco-2 cells and 10% for HEK-293 cells). Caco-2 cells were plated at a density of 1 x 10⁵ cells/cm² and either transfected while still in suspension or left untransfected for RNA extraction. After 24 h, cells were incubated with 25-hydroxycholesterol (25-HCH) or Mevinolin for an additional 24 h in media containing serum treated with 1% charcoal, whereas control cells were treated with ethanol alone (vehicle). In other experiments, cells were transiently cotransfected with NPC1L1 promoter construct and SREBP-2 expression vector by electroporation utilizing Amaxa technology (Amaxa) and plated at the same density as mentioned above. 25-HCH and Mevinolin were both obtained from Sigma (St. Louis, MO).

RNA extraction and real-time RT-PCR analysis. Total RNA was prepared from Caco-2 cells treated as indicated above using an Absolutely RNA RT-PCR Miniprep kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Equal amounts of RNA from both treated and control samples were reverse transcribed and amplified in a one-step reaction utilizing a Brilliant SYBR Green QRT-PCR Master Mix kit (Stratagene). Real-time PCR was performed using Mx3000P (Stratagene). hNPC1L1 was amplified with gene-specific primers (4) (sense primer: 5'-TATCTTCCCTGGTTCCTGAACGAC-3' and antisense primer: 5'-CCGCAGAGCTTCTGTGTAATCC-3'). -Actin was amplified as an internal control using gene-specific primers (sense primer: 5'-CATGTTTGAGACCTTCAACAC-3' and antisense primer: 5'-CCAGGAAGGAAGGCTGGAA-3'). Since the amplification efficiencies for both NPC1L1 and -actin were approximately equal, quantitation was expressed as the ratio of 2^C_T– NPC1L1/2^C_T– -actin, where C_T – NPC1L1 and C_T – -actin represent the differences between the threshold cycles (C_T) of amplification of treated and control RNA for NPC1L1 and -actin, respectively.

Plasmid construction. Three different fragments from the promoter region of hNPC1L1 were amplified utilizing human genomic DNA (Promega, Madison, WI) and gene-specific primers based on the sequence deposited in gene bank. Three different forward primers and a reverse primer were used in the PCRs to amplify different fragments representing 5' deletions of the NPC1L1 promoter. Forward primers contained an internal site for the KpnI restriction enzyme (underlined), and their sequences were as follows: primer 1, 5'-GGGGTACCTGGACTCTATCTCTCTGTGG-3'; primer 2, 5'-GGGGTACCCCACTATGGCTGTCTTGAGA-3'; and primer 3: 5'-GGGGTACCGGTCCCATCTGTGCCTCCAG-3'. The sequence of the reverse primer contained a site for the BglII enzyme (underlined) and was 5'-GGAAGATCTCCCAGGTCTGGGAAGGGGTCA-3'. Amplifications were performed using a proof-reading Elongase enzyme mix (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. PCR products were then digested with KpnI and BglII enzymes and subcloned into a luciferase reporter gene vector, pGL-2 basic (Promega). The fidelity of the constructs was then confirmed by sequencing, and plasmids were prepared for transfection using a kit from Qiagen (Valencia, CA). Single-point mutations in the NPC1L1 promoter construct were made using the QuikChange Site-Directed Mutagenesis kit from Stratagene according to the manufacturer’s instructions. The forward sequences of the primers used were as follows: MSREN1, 5'-GCTGTGTCATCGAAAAAGAAGAGGCTGCCTTAATG-3' for mutations introduced in the stearol response element of NPC1Ll promoter (SREN1) cis-element; and MSREN2, 5'-GTCCCTTGCTAAAGATGACCGGTGGGA-3' for mutations introduced in the SREN2 cis-element (mutated points are underlined).

Transient transfection and luciferase assay. For transfection studies, Caco-2 cells (2 x 10⁵) were seeded into 24-well plates and cotransfected while still in suspension with one of the hNPC1L1 promoter-luciferase constructs and pCMV, a -galactosidase mammalian expression vector (BD Biosciences Clontech, Palo Alto, CA) using Lipofectamine 2000 reagent (Invitrogen). The latter plasmid served as an internal control for transfection efficiency. A total of 3 µg DNA/well, at a ratio of 5:1 for experimental versus pCMV, was used for each transfection. In some experiments, Caco-2 cells were transfected utilizing an Amaxa Nucleofector System according to the manufacturer’s instructions. Briefly, 2 x 10⁶ cells were harvested and then electroporarted in 100 ml of solution T (supplied by Amaxa) along with 8 µg of NPC1L1 promoter construct, 2 µg of pCMV, and various amounts of mammalian expression vectors for SREBPs that were obtained from ATCC. Cells were then transferred to full media and plated on 8 wells of a 24-well plate. After 24 h, cells were washed with PBS and lysed using a kit from Promega. The activities of both firefly luciferase and -galactosidase were measured by a luminometer according to the manufacturer’s instructions from kits obtained from Promega and Clontech, respectively. Promoter activity was expressed as the ratio of luciferase to -galactosidase activity in each sample.

Nuclear protein extraction and gel mobility shift assay. Nuclear extracts from Caco-2 cells transfected with SREBP expression vectors were prepared using NE-PER nuclear and cytoplasmic extraction reagents from Pierce (Rockford, IL) according to the manufacturer’s instructions. Double-stranded oligonucleotides were end labeled with T4-polynucleotide kinase and [³²P]ATP (Amersham) and utilized as probes for gel mobility shift assays (GSAs). The sequences of the probes were as follow: SREN1 (–45/–13 bp), 5'-TCGAGTCATCGAAGGGGAGGAGGCTGCCTTAAT-3'; SREN2 (–667/–639 bp), 5'-TCGACCCTTGCTAGGGGTGACCGGTGGGA-3'; and LDL receptor (LDLR), 5'-TCGAGATCAAAATCACCCCACTGC-3'.

DNA-protein binding reactions were performed as previously described (18). Briefly, reactions in 20 µl total were initiated by adding 20,000 counts/min of the probe to 10 µg of nuclear extract in binding buffer containing 50 mM Tris·HCl (pH 7.5), 1 mM MgCl₂, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 1 µg/sample poly(dI-dC)·(dI-dC), and 5% (vol/vol) glycerol. Reactions were maintained at room temperature for 30 min prior to electrophoresis. DNA-protein complexes were resolved on a 6% polyacrylamide gel in buffer containing 90 mM Tris, 90 mM boric acid, and 2 mM EDTA. Gels were dried and subjected to autoradiography at –80°C.

Statistical analysis. Results are expressed as means ± SE. Student’s t-test was utilized for statistical analysis. A P value of 0.05 or less was considered statistically significant.

	RESULTS

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Modulation of NPC1LI mRNA expression by sterols. A recent study (5) has demonstrated a reduction in NPC1L1 expression in mice fed with a cholesterol-enriched diet. Oxysterols, such as 25-HCH, are naturally occurring intermediates of cholesterol metabolism that have been previously shown to regulate the expression of intestinal proteins involved in cholesterol homeostasis (1, 20, 25). Therefore, we studied the effect of 25-HCH on intestinal hNPC1L1 expression. Caco-2 cells were incubated with different concentrations of 25-HCH, and levels of NPC1L1 mRNA were assessed by real-time RT-PCR. PCR amplification utilizing hNPC1L1 and -actin (internal control) gene-specific primers clearly showed that NPC1L1 mRNA levels in Caco-2 cells were significantly decreased by 24 h of incubation with 25-HCH in a dose-dependent manner, with maximal inhibition occurring at a concentration of 5 µg/ml (Fig. 1A). These data indicated a reduction in NPC1L1 expression in response to sterol addition in human intestinal epithelial cells. We further evaluated the effect of cholesterol depletion on NPC1L1 expression. Cellular cholesterol depletion was achieved, as previously described (25), by incubating Caco-2 cells with Mevinolin, which inhibits cellular cholesterol synthesis by blocking the activity of 3-hydroxy-3-methylglutaryl-CoA reductase. As shown in Fig. 1B, incubation with 40 µM Mevinolin significantly increased levels of NPC1L1 mRNA in Caco-2 cells. Overall, these findings strongly suggest that NPC1L1 expression is subject to regulation by cholesterol and its derivative 25-HCH in intestinal Caco-2 cells.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Human Niemann-Pick C1-like 1 (hNPC1L1) mRNA expression is altered by cholesterol. Caco-2 cells were incubated for 24 h with different concentrations of 25-HCH (A) or Mevinolin (B) in medium containing 1% charcoal-treated serum. RNA was extracted, and real-time RT-PCR was performed utilizing SYBR green fluorescent dye as described in METHODS. Levels of hNPC1L1 mRNA were normalized to levels of human -actin mRNA and expressed as percentage of control. Data are shown as means ± SE of values obtained from at least 3 different experiments performed on separate occasions. *P < 0.05 compared with control.

View larger version (8K): [in this window] [in a new window]   Fig. 2. hNPC1L1 is highly active in Caco-2 cells. The promoter fragment (–1741/+56, where +1 represents the transcription initiation site) of the hNPC1L1 gene was amplified from human genomic DNA and then cloned into the pGL2 promoterless reporter vector. Both human intestinal Caco-2 and human embryonic kidney (HEK)-293 cells were transiently cotransfected with the hNPC1L1 promoter construct along with the pCMV vector. Relative promoter activity was measured by the firefly luciferase assay and normalized to -galactosidase activity to correct for transfection efficiency. The activity of the promoter is expressed as the fold increase compared with the pGL2 empty vector alone. Results were obtained from 3 separate experiments and are expressed as means ± SE. *P < 0.05 compared with empty vector.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Effect of cholesterol on hNPC1L1 promoter activity. Caco-2 cells were transiently cotransfected with the hNPC1L1 promoter fragment (–1741/+56) and pCMV. Twenty-four hours posttransfection, cells were incubated with 5 µg/ml 25-hydroxycholesterol (25-HCH; A) or 40 µM Mevinolin (B) for an additional 24 h. C: Caco-2 cells were transfected with different hNPC1L1 promoter fragments representing progressive 5' deletions of the promoter and then treated with Mevinolin as described in B. Cells were then harvested, and promoter activity was measured by the luciferase assay. Values were normalized to -galactosidase to adjust for transfection efficiency. Data were obtained from at least 3 different experiments performed at 3 separate occasions and are shown as means ± SE. Results are expressed as percentages of control compared with cells treated with vehicle alone.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Sterol response element (SRE) binding protein (SREBP)-2 transactivates hNPC1L1 promoter activity and expression. A: Caco-2 cells were cotransfected with hNPC1L1 promoter construct and expression vectors for SREBP-2, SREBP-1a, or SREBP-1c along with the pCMV vector. Data were obtained from 3 separate experiments and are expressed as means ± SE. B: Caco-2 cells were transiently transfected with SREBP-2 for 48 h. Cells were then harvested, and total RNA was extracted. Real-time PCR was performed using gene-specific primers for hNPC1L1 and -actin (internal control). Levels of hNPC1L1 mRNA were normalized to levels of -actin and expressed as percentages of control. *P < 0.05 compared with control.

View larger version (16K): [in this window] [in a new window]   Fig. 5. SRE cis-elements of hNPC1L1 promoter binding to SREBP-2. A: sequence alignment between the complement of SRE cis-element of the LDL receptor (LDLR) gene and SREN1 and SREN2 cis-elements of the hNPC1L1 gene. B: nuclear extracts from cells overexpressing SREBP-2 were incubated with the 32P-labeled SRE cis-element of the promoter of LDLR. Protein-DNA complexes competed in the presence of excess of cold SRE-LDLR (lane 3) or the presence of excess of SREN1 of the hNPC1L1 promoter (lane 4). Excess of oligonucleotides representing the activator protein (AP)-1 consensus sequence failed to compete with the binding (lane 5). C: nuclear extracts were incubated with 32P-labeled SREN1 or 32P-labeled SREN2 of the hNPC1L1 promoter. Complexes were competed by excess of unlabeled probes (lanes 3 and 7) and in the presence of excess of unlabeled SRE-LDLR. Gels shown are representative of at least 3 separate experiments with similar results.

SREBP-2 is involved in cholesterol-mediated modulation of the NPC1L1 promoter. To further investigate the role of SREBP-2 in the alterations of the NPC1L1 promoter by cholesterol, we examined the effect of sterols on hNPC1L1 promoter constructs harboring mutations in SREN1 and SREN2 sites. As shown in Fig. 6A, the basal activity of the promoter was not altered by a mutation in the distal SREN2 site. Also, the construct harboring a mutation only in the SREN2 site demonstrated a lesser but significant response to both Mevinolin and 25-HCH (Fig. 6, A and B). This retained response to sterols is likely attributable to an intact SREN1 cis-element. However, the data depicted in Fig. 6, A and B, show that the mutation in the SREN1 cis-element alone significantly reduced the basal activity of the promoter and abrogated the response to either Mevinolin or 25-HCH. These data clearly demonstrate that the SREN1 cis-element is essential for driving the basal activity of the hNPC1L1 promoter as well as its regulation by sterols, whereas the SREN2 cis-element appears to be involved only in the modulation of the hNPCL1 promoter in response to sterols.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Mutations in SRE cis-elements attenuate the response of hNPC1L1 promoter to sterols and SREBP-2. Caco-2 cells were transiently transfected with the hNPC1L1 promoter fragment, a fragment with a mutated SREN1 site (mutant sites are shown as white boxes) or mutated SREN2 site, or a fragment harboring mutations in both the sites along with the pCMV vector (A and B). The relative promoter activity was expressed as arbitrary units (A) in means ± SE of values obtained from 3 separate experiments. Controls were normalized to 100% in B. Cells were treated after 24 h with either 40 µM Mevinolin or 5 µg/ml 25-HCH for an additional 24 h. hNPC1L1 promoter activity was then assessed and normalized to -galactosidase values as described in Fig. 4. Data were obtained from 3 separate experiments presented as percentages of control and expressed as means ± SE. *P < 0.05 compared with control.

	DISCUSSION

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It is noteworthy to mention that the promoter of NPC1L1 showed high activity in human intestinal Caco-2 cells, consistent with the fact that NPC1L1 is predominantly expressed in the small intestine (2, 4). Also, a recent study (4) has demonstrated the expression of NPC1L1 in Caco-2 cells, indicating their suitability as in vitro cellular model for enterocytes to investigate the molecular regulation of NPC1L1 expression. Our study also suggested that hNPC1L1 promoter activity is directly regulated by oxysterol and cholesterol depletion, corresponding with parallel alterations in the level of its mRNA expression. It should be noted that the concentrations of both 25-HCH and Mevinolin used in the present experiments are within the range of previously used concentrations to investigate the effect of sterol addition and depletion on the expression of various genes in different cell culture models including Caco-2 cells (8, 13, 14, 25). Also, examination of cells with a light microscope revealed that treatment with either 25-HCH or Mevinolin did not inflict any damage to the cells (data not shown).

Three fragments representing progressive deletions in the promoter exhibited similar basal activity, indicating that the region flanking the area between –291 and +56 nt (the smallest fragment, NPC1L1F3) harbors cis-elements sufficient to drive the basal activity of the promoter in Caco-2 cells. Importantly, cholesterol depletion significantly stimulated the three fragments of the NPC1L1 promoter but with different magnitudes. The activity of both NPC1L1F1 and F2 fragments (–1741/+56 and –1091/+56) were similarly induced (by 2 fold), whereas the NPC1L1F3 fragment was only stimulated by 33% in response to Mevinolin. These findings indicated the presence of potential cholesterol response elements in the region flanking the area between –1091 and +59 nt in the NPC1L1 promoter.

In general, oxysterols elicit their effects via the induction of several pathways that influence the transcription of various genes. These pathways include the activation of the orphan nuclear receptor LXR (17), binding to oxysterol binding protein (20), and downregulation of SREBP (11), which are major regulators of the transcription of genes involved in cholesterol homeostasis (7). Our data, which clearly showed the decrease in NPC1L1 promoter activity by 25-HCH and its induction by cholesterol depletion, indicated that SREBPs are involved in the observed modulation of NPC1L1 expression by cholesterol.

SREBPs belong to a family of transcription factors that contain a basic helix-loop-helix-zip domain and consists of three isoforms designated as SREBP-1a, SREBP-1c, and SREBP-2, which play different roles in the pathways of cholesterol and lipid synthesis (7, 10–12). The transcriptional activity of SREBPs is influenced by cholesterol by proteolytic modifications (7). Low levels of cholesterol result in a cleavage of the NH₂-terminus of inactive SREBPs, producing the mature transcription factor, which translocates to the nucleus to affect gene expression (11). This proteolytic process is inhibited by high levels of cholesterol (11). This sterol-dependent activation of SREBPs has been previously shown to be present in human intestinal Caco-2 cells (8), further lending support to their potential involvement in the observed regulation of NPC1L1 by sterols.

Our study provides novel evidence for the role of SREBP-2 in the regulation of NPC1L1 promoter activity. Interestingly, we found SREBP-2 to be the major regulator since its effect on the NPC1L1 promoter was markedly higher (50-fold) compared with the effects of SREBP-1a and -1c. Previous studies (7, 11) have suggested that SREBP-1c is implicated in the regulation of fatty acid synthesis, whereas SREBP-2 has been shown to be crucial for controlling cholesterol synthesis. The effects of SREBP-1a overlap, to a limited extent, with the regulatory roles of both SREBP-1c and SREBP-2 (7). Furthermore, the expression of intestinal SREBP-2 but not SREBP-1a or -1c was downregulated in mice fed with a cholesterol-enriched diet (25). Therefore, it is logical to propose that the intestinal SREBP-2 isoform is the key modulator of intestinal NPC1L1 expression.

To further analyze the role of SREBP-2, we investigated its binding to the hNPC1L1 promoter. Indeed, sequence analysis identified two cis-SREs flanking the region of –35/–26 bp (proximal SRE or SREN1) and the region of –657/–648 (distal SRE or SREN2). Both regions share high homology (50% and 70%, respectively) with a previously described SRE cis-element on the promoter of LDLR (23). Mutations in each of the SRE cis-elements attenuated the transactivation of the promoter as well as the effect of Mevinolin, whereas double mutations completely abolished the response. Additionally, GSA demonstrated the binding of both SREN1 and SREN2 to SREBP-2 that was competed out with the LDLR SRE cis-element. These data provide strong evidence for the involvement of SREBP-2 in the regulation of NPC1L1 promoter activity by cholesterol. Interestingly, the mutation in SREN2 alone attenuated the response to sterol but was not sufficient to abolish it. However, the mutation of SREN1 alone resulted in a remarkable reduction in the basal activity of the promoter and completely abrogated its regulation by sterols. Taken together, these observations indicate that the SREN1 cis-element on the NPC1L1 promoter is essential for both its basal activity and regulation by SREBP-2, whereas the SREN2 cis-element appears to play a role only in the regulation of the hNPC1L1 promoter by SREBP-2. In view of the central role of NPC1L1 in intestinal cholesterol absorption (2, 4, 16), it is not surprising that its expression is under tight regulation by SREBP-2, having at least two cis-SREs in the 5' regulatory region of its gene.

It should be noted that the intestinal SREBP-2-dependent pathway was previously implicated as an adaptive intestinal process in response to high cholesterol by modulating the expression and activity of the intestinal apical sodium-dependent bile acid transporter (ASBT) (1, 21). The present data also suggest that the same adaptive mechanism is involved in the regulation of intestinal cholesterol absorption. Accordingly, it is proposed that a low level of cholesterol activates the transcriptional activity of intestinal SREBP-2, which, in turn, increases NPC1L1 and ASBT expression with parallel increases in intestinal cholesterol and bile acid absorption to restore cholesterol pool in the body. High cholesterol, however, leads to suppression in the activity of intestinal SREBP-2 and a subsequent reduction in the expression of both NPC1L1 and ASBT, resulting in a concomitant decrease in cholesterol and bile acid absorption favoring a decline in plasma cholesterol. It is plausible to propose that insufficiency in the response of intestinal SREBP-2 to high cholesterol may underlie the variations between human individuals in their responses to a high-cholesterol diet and the development of hypercholesterolemia.

Although a recent study (6) suggested a role for LXR nuclear factors in the regulation of hNPC1L1 expression, there is no direct evidence for the activation of the NPC1L1 promoter by LXR. Future studies will focus on investigating the role of other transcription factors such as LXR in the regulation of NPC1L1 promoter activity by cholesterol and determine the mechanisms of possible cross-talk between LXR and SREBP-2 pathways. Since disturbances in cholesterol homeostasis lead to elevated serum cholesterol levels, an important risk factor for cardiovascular diseases, diabetes, and obesity, these studies will expand our knowledge and enhance our ability to manage such cholesterol-related disorders.

In summary, our present study provides novel evidence for the involvement of SREBP-2 in the regulation of intestinal hNPC1L1 expression. We also identified two putative SRE cis-elements in the hNPC1L1 promoter and demonstrated the involvement of these elements in mediating the modulation of NPC1L1 promoter activity by cholesterol.

	GRANTS

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This work was supported by the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-71596 (to W. A. Alrefai), DK-54016 (to P. K. Dudeja), and DK-68324 (to P. K. Dudeja).

	FOOTNOTES

 

Address for reprint requests and other correspondence: W. A. Alrefai, Univ. of Illinois and Jesse Brown Veterans Affairs Medical Center, Medical Research Service (600/151), 820 S. Damen Ave., Chicago, IL 60612 (e-mail: walrefai{at}uic.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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