Identification of a Functional Vitamin D Response Element in the Murine Insig-2 Promoter and Its Potential Role in the Differentiation of 3T3-L1 Preadipocytes
Seunghee Lee,
Dong-Kee Lee,
Eunho Choi and
Jae W. Lee
Division of Diabetes, Endocrinology and Metabolism, Department of Medicine (D.-K.L., J.W.L.), Department of Molecular & Cellular Biology (S.L., J.W.L.), Baylor College of Medicine, Houston, Texas 77030; and Department of Life Science (E.C.), Pohang University of Science and Technology, Pohang 790-784, Korea
Address all correspondence and requests for reprints to: Jae W. Lee, Ph.D., Department of Medicine, Division of Diabetes, Endocrinology & Metabolism, Dept. Molecular & Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: jwlee{at}bcm.tmc.edu.
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ABSTRACT
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Insulin-induced gene-1 (Insig-1) and its homolog Insig-2 encode closely related proteins of the endoplasmic reticulum that block proteolytic activation of sterol regulatory element binding proteins, membrane-bound transcription factors that activate synthesis of cholesterol and fatty acids in animal cells. These proteins also restrict lipogenesis in mature adipocytes and block differentiation of preadipocytes. Herein, we identified a novel 1
,25-dihydroxyvitamin D3 [1,25-(OH)2D3] response element in the promoter region of Insig-2 gene, which specifically binds to the heterodimer of retinoid X receptor and vitamin D receptor (VDR) and directs VDR-mediated transcriptional activation in a 1,25-(OH)2D3-dependent manner. Interestingly, 1,25-(OH)2D3 is known to directly suppress the expression of peroxisome proliferator-activated receptor
2 protein and inhibits adipocyte differentiation of 3T3-L1 preadipocytes and murine bone marrow stromal cells. Consistent with an idea that the antiadipogenic action of 1,25-(OH)2D3 may also involve up-regulation of Insig-2, we found that 1,25-(OH)2D3 transiently but strongly induces Insig-2 expression in 3T3-L1 cells. This novel regulatory circuit may also play important roles in other lipogenic cell types that express VDR, and collectively our results suggest an intriguing, new linkage between 1,25-(OH)2D3 and lipogenesis.
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INTRODUCTION
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STEROL REGULATORY ELEMENT-BINDING proteins (SREBPs) control synthesis of lipids in liver, adipocytes, and other organs. Synthesized on membranes of the endoplasmic reticulum (ER), these proteins translocate to the Golgi complex, where they are processed sequentially by two proteases (1, 2). These cleavages release a functional transcription factor that enters the nucleus and activates genes that produce a variety of enzymes required for the synthesis of cholesterol and unsaturated fatty acids as well as phospholipids and triglycerides. Notably, SREBP-1a activates both the cholesterol and fatty acid biosynthetic pathways. In contrast, SREBP-1c and SREBP-2, the predominant isoforms in liver, are relatively specific for fatty acid and cholesterol biosyntheses, respectively. SREBP-1c also regulates adipocyte differentiation (1, 2).
The activities of the SREBPs are known to be controlled at both transcriptional and posttranscriptional levels. For instance, transcription of the SREBP-1c gene is enhanced markedly by insulin and suppressed by glucagon in the liver (3, 4, 5). The insulin-mediated enhancement of SREBP-1c gene transcription provides a mechanism by which insulin increases the synthesis of fatty acids in the liver. It is also noted that the SREBP-1c and SREBP-2 gene is activated by nuclear SREBPs in a feed-forward fashion (6). Posttranscriptionally, SREBP activity is regulated primarily by sterols, which inhibit the proteolytic processing of the membrane-bound SREBP precursors (1, 2). This control is mainly mediated by SREBP cleavage activating protein (SCAP) (7) and proteins encoded by insulin-induced gene-1 (Insig-1) and its homolog Insig-2 (8, 9, 10). Newly synthesized SREBPs form tight complexes with SCAP, a polytopic membrane protein of the ER. With conditions of high sterol demand, the SCAP/SREBP complex is incorporated into vesicles that bud from the ER and travel to the Golgi, where SREBP processing occurs (11). With sterol excess, the SCAP/SREBP complex binds to Insigs, intrinsic membrane proteins of the ER (8, 9). This binding prevents the SCAP/SREBP complex from being incorporated into transport vesicles. Thus, SREBPs remain trapped in the ER, and proteolytic processing is blocked. The nuclear content of SREBPs declines rapidly as a result of proteasomal degradation, and accordingly the synthesis of cholesterol and fatty acids declines.
Insig-1 and Insig-2 proteins are 59% identical, and both bind SCAP in a sterol-dependent fashion (8, 9). These two proteins differ, however, in their mode of regulation. In cultured cells, transcription of the Insig-1 gene requires nuclear SREBPs, and this transcription declines markedly when SREBP activity is down-regulated. In contrast, Insig-2 activity in cultured cells appears to be constitutive and does not require nuclear SREBPs. This divergent regulation amplifies feedback control. When nuclear SREBP activity is low, Insig-2 is the only form of Insigs present in the cell. If intracellular levels of cholesterol fall, SREBP cleavage can be activated rapidly. When SREBPs enter the nucleus, they increase the amount of Insig-1 mRNA, and this sensitizes the system to inhibition when sterol levels rise (8, 9). Insig-2a is a recently identified liver-enriched transcript from the Insig-2 gene (12). This transcript and the ubiquitous transcript, designated Insig-2b, differ through the use of different promoters that produce different noncoding first exons that splice into a common second exon. Although the Insig-2a and -2b mRNAs encode identical proteins, they differ in patterns of regulation. Insig-2a is the predominant transcript in livers of fasted animals, and it is selectively down-regulated by insulin. Thus, Insig-2a mRNA increases when mice are fasted, and it declines when they are refed. The transcript also increases in livers of rats whose insulin-secreting pancreatic ß-cells have been destroyed by streptozotocin, and it is reduced when insulin is injected. The insulin-mediated fall in Insig-2a may allow SREBP-1c to be processed, thereby allowing insulin to stimulate fatty acid synthesis, even under conditions in which hepatic cholesterol levels are elevated (12).
Some members of the nuclear receptor superfamily act as metabolic and toxicological sensors, enabling the organism to quickly adapt to environmental changes by inducing the appropriate metabolic genes and pathways (13, 14). Ligands for these metabolic receptors include compounds from dietary origin, intermediates in metabolic pathways, drugs or other environmental factors that, unlike classical nuclear receptor ligands, are present in high concentrations. These receptors integrate the homeostatic control of energy and glucose metabolism through peroxisome proliferator-activated receptor (PPAR)
, fatty acid, triglyceride and lipoprotein metabolism via PPARs, reverse cholesterol transport and cholesterol absorption through the liver X receptors (LXRs) and liver receptor homolog-1, bile acid metabolism through the farnesol X receptor (FXR), LXRs and liver receptor homolog-1, and the defense against xeno- and endobiotics by the pregnane X receptor/steroid and xenobiotic receptor and constitutive androstenol receptor (13, 14). Accordingly, altered signaling by these receptors from either chronic ligand excess or genetic mutations may cause an imbalance in these homeostatic circuits, and contribute to the pathogenesis of common metabolic diseases such as obesity, insulin resistance and type 2 diabetes, hyperlipidemia and atherosclerosis, and gallbladder disease.
The vitamin D receptor (VDR), which mediates the effects of the calcemic endocrine hormone 1
,25-dihydroxyvitamin D3 [1,25-(OH)2D3], was recently shown to function as a receptor for the secondary bile acids such as lithocholic acid (LCA) (15), which are hepatotoxic and potential enteric carcinogens. VDR is an order of magnitude more sensitive to LCA and its metabolites than are other related nuclear receptors. Notably, activation of VDR by LCA or 1,25-(OH)2D3-induced expression of CYP3A, a cytochrome P450 enzyme that detoxifies LCA in the liver and intestine. Although the physiological significance of this observation is not yet well established, it offers a mechanism that may explain the proposed protective effects of 1,25-(OH)2D3 and its receptor against colon cancer (15). 1,25-(OH)2D3 is also well known for its activity to inhibit adipocyte differentiation of 3T3-L1 preadipocytes and murine bone marrow stromal cells, likely through directly repressing the expression of the potent adipogenic transcription factor PPAR
2 (16, 17, 18, 19).
Interestingly, Insig-1 and Insig-2 have recently been shown to restrict lipogenesis in mature adipocytes and block differentiation of preadipocytes (20). Hepatic Insig-1 or -2 overexpression was also shown to reduce lipogenesis in obese Zucker diabetic fatty rats and in fasted/refed normal rats (21). Similarly, overexpression of Insig-1 in the livers of transgenic mice was demonstrated to inhibit SREBP processing and reduces insulin-stimulated lipogenesis (22). In this report, we discovered that 1,25-(OH)2D3 strongly induces Insig-2a expression in 3T3-L1 cells, and accordingly identified a functional 1,25-(OH)2D3 response element in the promoter region of Insig-2a gene. These results suggest an interesting linkage between the antiadipogenic action of 1,25-(OH)2D3 (16, 17, 18, 19) to up-regulation of antilipogenic protein Insig-2 (20, 21, 22). This novel regulatory circuit may also block further lipid synthesis in other VDR-expressing lipogenic cells in which SREBPs play essential roles.
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RESULTS AND DISCUSSION
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Identification of VDR Response Element (VDRE) in the Insig-2 Promoter Region
In our recent cDNA microarray analyses, we found murine hepatic Insig-2 as a putative LXR target gene, which was up-regulated by approximately 4-fold in the presence of LXR ligand T0901317 (23). While reexamining these results in a variety of different hepatic cell lines, we were surprised to find that Insig-2 expression is significantly enhanced by 1,25-(OH)2D3 and LCA as well as several other ligands that were previously shown to lower cholesterol and triglyceride levels (data not shown). Because LCA and other related secondary bile acids were recently shown to serve as additional ligands for VDR along with 1,25-(OH)2D3 (15), the results with 1,25-(OH)2D3/LCA could result from direct binding of VDR to the Insig-2 promoter region. Thus, we searched for direct repeat 3 (DR3)-type elements known to bind VDR (24) as well as DR4-type elements that may mediate the modulatory function of LXR ligand T0901317 (25). Throughout the first intron (located between the first exon E1a and the alternative first exon E1b) as well as the upstream regions from E1a, which likely include the core promoter and other important regulatory elements, computer analysis identified at least three DR3 and nine DR4-like sequences (Fig. 1A
). To test whether any of these elements is responsible for either our reported T0901317/LXR effect on Insig-2 expression (23) or the unexpected effects of 1,25-(OH)2D3 and LCA, we inserted three different Insig-2 genomic regions encompassing these elements into thymidine kinase (TK) minimal promoter-luciferase reporter constructs. Fragment I contains DR41 and 2 and DR31 and 2. Fragments II and III contain DR43 and DR44 to 9, respectively. Fragment III also contains DR33 (Fig. 1A
). Interestingly, none of these fragments directed LXR-dependent transactivation in HepG2 cells, and none was active with FXR either (Fig. 1B
). However, fragment I directed approximately 23-fold induction by 1,25-(OH)2D3, whereas fragments II and III were without any significant effect (Fig. 1B
). It should be noted that this activation is VDR dependent because HepG2 cells showed almost no 1,25-(OH)2D3 response without coexpressed VDR (data not shown). Overall, these results suggest the presence of functional VDRE within the upstream region of Insig-2a, whereas the nature of LXR-mediated induction of Insig-2 (23) still remains unresolved.

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Fig. 1. Identification of VDRE in the Insig-2 Promoter
A, Schematic representation of the Insig-2 promoter region. The first two alternative exons E1a and E1b, DR-4 elements (open), DR3 elements (closed) and fragments I, II, and III are as shown. The first base in the exon E1a was arbitrarily assigned as +1, and the upstream and downstream sequences from this first base were indicated as and +, respectively. B, Indicated luciferase reporter constructs were cotransfected into HepG2 cells, along with LacZ expression vector (100 ng) and expression vectors (10 ng) for LXR, FXR and VDR, as indicated. Closed and shaded boxes indicate the absence and presence of ligand (1 µM T0901317, 10 µM CDCA, and 0.01 µM 1,25-(OH)2D3), respectively. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions.
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To precisely map the functional VDRE, we performed further deletional studies. TK-luciferase reporter constructs directed by fragments Ia, Ib, and Ic (Fig. 2A
) were constructed and tested in cotransfections of HepG2 cells. As shown in Fig. 2B
, Ib and Ic but not Ia responded to 1,25-(OH)2D3. Although Ic showed significantly lower activities than Ib, the fold activations were similar to each other because the basal activities of fragment Ic was also lower. These results suggest that the region between Ib and Ic may contain a negative regulatory element that suppresses both the basal and induced levels of transactivation. Our computer analysis also revealed the presence of multiple DR1 and DR2/5 type elements within the fragment I (data not shown), which serve as response elements of PPARs and retinoic acid receptors (RARs), respectively (13, 14). However, fragment I did not respond to PPAR
and RAR ligands (Fig. 2C
). Interestingly, fragment I responded to 1,25-(OH)2D3 very poorly in HEK293 cells, whereas fragments Ib and Ic were completely functional, suggesting the presence of a negative regulatory element within the 3184/2372 region that binds a factor/factors present in HEK293 cells (our unpublished results). Overall, these results strongly support the presence of a putative VDRE in the region between 3472 and 3340.

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Fig. 2. Deletion Analyses of Insig-2 Fragment I
A, Schematic representation of the Insig-2 fragment I deletion mutants. Three DR-4 elements (open) and two DR3 elements (closed) are as shown. B and C, Indicated luciferase reporter constructs were cotransfected into HepG2 cells, along with LacZ expression vector (100 ng) and expression vectors (10 ng) for VDR, RAR , and PPAR , as indicated. Closed and shaded boxes indicate the absence and presence of ligand (0.01 µM 1,25-(OH)2D3, 0.1 µM 9-cis-RA, and 1 µM rosiglitazone), respectively. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions.
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As noted already, the fragment I sequences contain at least two prominent DR3-type elements located at 3379/3365 and 2484/2470, respectively (Fig. 3A
). The 2484/2470 sequences are more closely related to the consensus DR3 (designated as Insig-2 DR3), whereas the sequences at 3379/3365 are highly homologous to the previously defined VDRE from the human osteocalcin gene (26) (designated as Insig-2 VDRE). In particular, the homology includes the highly conserved G in the space region, which appears to be important in VDR binding (24, 26). To test their functionality, we have inserted three copies of oligonucleotides derived from these two elements into the upstream region of TK-Luc reporter, respectively. In cotransfection of HepG2 cells, three copies of Insig-2 VDRE mediated over 72-fold induction with 1,25-(OH)2D3 (Fig. 3B
), whereas three copies of Insig-2 DR3 were inert. Notably, these results nicely correlate with the deletional studies (Fig. 2
). Consistent with the notion that this Insig-2 VDRE is a sole major determinant for the 1,25-(OH)2D3-dependent transactivation of the Insig-2 promoter fragment I, introduction of specific mutation into the Insig-2 VDRE (i.e. TGCCCt Cgt TacCCt to TGCaat agt Tacaat, in which lower case letters indicate bases that deviate from the consensus sequences; underlined letters indicate those mutated) completely abolished the 1,25-(OH)2D3-response observed with the Insig-2 fragment I (i.e. see the results with Im-TK-Luc in Fig. 3B
). Consistent with the recent report that VDR also serves as a specific receptor for secondary bile acids such as LCA (15), similar results were also obtained with LCA (Fig. 3C
). In addition, a mutant VDR-Y236A that specifically lacks coactivator bindings (27) acted as a dominant- negative mutant of transactivation directed by either fragment I or three copies of Insig-2 VDRE (Fig. 3D
). Overall, these results suggest that the Insig-2 VDRE is a genuine VDRE, which may directly bind VDR to confer the 1,25-(OH)2D3/LCA-dependent transactivation response to the Insig-2 gene.

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Fig. 3. Fine-Mapping of Insig-2 VDRE
A, Schematic representation of three additional reporter constructs are as shown, in which three copies of Insig-2 DR3 and VDRE as well as Im, a fragment identical with Insig-2 fragment I except a specific mutation in its VDRE region were placed upstream of TK-Luc reporter. Osteocalcin VDRE and consensus VDRE (24 26 ) are as shown. BD, Indicated luciferase reporter constructs were cotransfected into HepG2 cells, along with LacZ expression vector (100 ng) and expression vector for VDR (10 ng) and/or VDR Y236A (10 ng), as indicated. Closed and shaded boxes (or and + in D) indicate the absence and presence of 0.01 µM 1,25-(OH)2D3 (B, D) or 100 µM LCA (C), respectively. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions.
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Direct Binding of VDR/Retinoid X Receptor (RXR) to the Insig-2 VDRE
To confirm that the Insig-2 VDRE is a direct biding site for the VDR/RXR heterodimer, we performed a gel mobility shift assay using 32P-labeled oligonucleotides encompassing the Insig-2 VDRE. As shown in Fig. 4A
, a shifted band was observed in the presence of both VDR and RXR but not each receptor alone, which was competed by an increasing amount of unlabeled, identical oligonucleotides but not by oligonucleotides specifically mutated in the VDRE region. This band was also abolished by VDR antibody that recognizes the DNA binding domain of VDR and disrupts the VDR-VDRE interactions (28) but not by a control antibody (Fig. 4A
). From these results, we concluded that the Insig-2 VDRE is a direct binding site for the VDR/RXR heterodimer.

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Fig. 4. VDR-Binding to Insig-2 VDRE
A, Gel-shift assays to study interactions of the VDR/RXR heterodimer with Insig-2 VDRE. Reticulocyte lysate (2 µl) containing in vitro-translated VDR and RXR were incubated alone or together with 32P-labeled Insig-2 VDRE, as indicated. The VDR antibody we used was previously described to abolish the VDR-VDRE interactions (28 ). The specific and nonspecific competitors are unlabeled Insig-2 VDRE probe and its mutated version in the VDRE region, respectively (1x, 10x, and 100x of the probe). B, Q-PCR analysis was performed to measure the relative amount of Insig-2a mRNA isolated from 3T3-L1 cells treated with an increasing amount of 1,25-(OH)2D3 and LCA for 24 h. Detection was performed by measuring SYBR Green binding to double-stranded DNA. Cyclophilin A mRNA was used as a reference, and the level of mRNA from untreated cells was arbitrarily assigned as 1. C, Insig2-fragment I-TK-Luc reporter (I-Luc) was cotransfected into HepG2 cells, along with LacZ expression vector (100 ng) and expression vector for VDR (10 ng), as indicated, and these cells were treated with an increasing amount of 1,25-(OH)2D3 and LCA for 24 h. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions.
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Induction of Insig-2a by 1,25-(OH)2D3 in 3T3-L1 Preadipocytes
The above results suggest an intriguing regulatory network in lipid homeostasis, in which 1,25-(OH)2D3 or increased level of secondary bile acids such as LCA induce Insig-2 expression, possibly shutting off further lipid synthesis in the liver and likely other organs in which SREBPs play essential roles. However, it is noted that VDR appears to be present in the liver only in a limiting amount (29). The human, rat, and mouse hepatocytes were shown to express very low VDR mRNA and protein levels, whereas sinusoidal endothelial, Kupffer, and stellate cells of normal rat livers as well as the mouse biliary cell line BDC and rat hepatic neonatal epithelial SD6 cells marginally expressed both VDR mRNA and protein (29). Kupffer, stellate, and endothelial cells also responded to 1,25-(OH)2D3 by a significant increase in the CYP24, indicating that the VDR in these cell types is fully functional (29). Thus, the VDR-mediated regulation of Insig-2 expression may play a role only in these selective hepatic cell populations, although their status of Insig-2 expression is currently unknown.
Based on the limiting expression status of VDR in hapatocytes (29), we suspected that the primary function of this newly discovered 1,25-(OH)2D3-mediated regulation of Insig-2 expression could be exerted in different tissues in which both VDR and Insig-2 are coexpressed. Interestingly, expression of Insigs was recently demonstrated to restrict lipogenesis in mature adipocytes and block differentiation in preadipocytes (20). The in vivo relevance of these findings was subsequently provided by two similar animal experiments. First, overexpression of hepatic Insig-1 or -2 reduced lipogenesis in obese Zucker diabetic fatty rats and in fasted/refed normal rats (21). Secondly, overexpression of Insig-1 in the livers of transgenic mice inhibited SREBP processing and reduced insulin-stimulated lipogenesis (22). Importantly, 1,25-(OH)2D3 was previously shown to suppress adipogenesis (16, 17, 18, 19) and VDR was reported to be induced during adipogenesis (30). Taken together, we suspected that the 1,25-(OH)2D3-mediated regulation of Insig-2 expression could provide an unexpected linkage between VDR and Insig-2 in adipogenesis.
To test this idea, we employed real-time quantitative PCR (Q-PCR) analyses with RNA isolated from 3T3-L1 preadipocytes treated with an increasing amount of 1,25-(OH)2D3 and LCA for 24 h. As shown in Fig. 4B
, Insig-2a was indeed induced by both of these two VDR-ligands in a dose-dependent manner. In particular, the concentration of 1,25-(OH)2D3 for the half-maximal activation of Insig-2a expression was estimated to be approximately 1 nM, although this value was hard to estimate for LCA due to its toxicity with concentration more than 100 µM (Fig. 4B
). We also measured the promoter activities of Insig2-fragment I-TK-Luc reporter from cotransfected HepG2 cells and obtained similar results; i.e. the concentration of 1,25-(OH)2D3 and LCA for the half-maximal activation was approximately 110 nM and 10 µM, respectively (Fig. 4C
).
Using RT-PCR analyses, we also extended these results to the expression pattern of Insig-2a, Insig-2b, an adipogenic marker aP2, VDR, and the loading control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during the adipogenic differentiation of 3T3-L1 preadipocytes (Fig. 5A
). 3T3-L1 preadipocytes were treated with the adipogenic differentiation mix for 2 d, which consists of 1 µM dexamethasone (Dex), MIX (3-isobutyl-1-methylxanthine, 0.5 mM), and insulin (10 µg/ml), and then with 10 µg/ml of insulin alone from d 3, as previously described (19). Under this condition, as demonstrated with oil red O staining, approximately 80% of these cells already exhibited small lipid droplets in their cytoplasm by the end of d 3, and most of the cells completed adipogenesis by d 56. As previously reported (19), treatment with 0.01 µM 1,25-(OH)2D3 or 10 µg/ml of insulin alone had no effect, whereas the differentiation mix-induced adipogenesis of 3T3-L1 cells was completely blocked in the presence of 0.01 µM 1,25-(OH)2D3 (data not shown). Consistent with these oil red O staining results, the induction of aP2 expression was significantly impaired in the presence of 1,25-(OH)2D3 (Fig. 5A
). The reported, transient induction pattern of VDR in the earlier phase of adipogenesis (30) was readily observed, and VDR was also induced by 1,25-(OH)2D3 alone in preadipocytes in support of the previously reported results (31). Importantly, 1,25-(OH)2D3 significantly induced the expression level of Insig-2a but not Insig-2b even in the presence of the adipogenic regimen (i.e. the differentiation mix/insulin). It should also be noted that the timing of this induction coincided with that of VDR; the level of both VDR and Insig-2a remains induced till d 2, and then rapidly diminished from d 3 (Fig. 5A
). It is important to note that Insig-2a mRNA was recently reported to lack in mature brown and white fats (12). Thus, Insig-2a mRNA could be only transiently induced by 1,25-(OH)2D3 during the early phase of adipogenesis (i.e. not in mature adipocytes) or preadipocytes. These results were independently confirmed in more quantitative real time Q-PCR analyses (Fig. 5B
). In particular, Insig-2b was barely induced by 1,25-(OH)2D3 (a maximum of 3-fold), whereas Insig-2a was dramatically induced by 1000- to 2800-fold (Fig. 5B
). Overall, these results strongly suggest that the 1,25-(OH)2D3-mediated regulation of Insig-2a expression likely plays an important role in 3T3-L1 cells and that the inhibitory action of 1,25-(OH)2D3 in adipogenesis (16, 17, 18, 19) might involve not only down-regulation of proadipogenic factor PPAR
2 (17) but also up-regulation of antiadipogenic protein Insig-2.

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Fig. 5. Induction of Insig-2a by 1,25-(OH)2D3 in Preadipocytes
A, RT-PCR analyses of 3T3-L1 cells during adipogenesis. RT-PCR was run against GAPDH, aP2, VDR, Insig-2b and Insig-2a. In the first set (indicated as Dex/MIX/Ins), 3T3-L1 cells were treated with the differentiation mix consisting of 1 µM Dex, MIX (3-isobutyl-1-methylxanthine, 0.5 mM) and insulin (10 µg/ml) for 2 d, and with 10 µg/ml of insulin from d 3. In the second set (indicated as 1,25-(OH)2D3), cells were treated with 0.01 µM 1,25-(OH)2D3 alone for 2 d, and with 0.01 µM 1,25-(OH)2D3 plus 10 µg/ml of insulin from d 3. In the last set of experiments (indicated as Dex/MIX/Ins + 1,25-(OH)2D3), cells were treated with the differentiation mix and 0.01 µM 1,25-(OH)2D3 for 2 d, and then with 0.01 µM 1,25-(OH)2D3 and 10 µg/ml of insulin from d 3. B, Q-PCR analysis was performed to measure the relative amount of each mRNA isolated from the above sets of cells. Detection was performed by measuring SYBR Green binding to double-stranded DNA. Cyclophilin A mRNA was used as a reference, and the level of 0 d mRNA was arbitrarily assigned as 1 for each gene.
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In this report, we identified a novel 1,25-(OH)2D3 response element in the promoter region of Insig-2 gene, which specifically binds to the RXR/VDR heterodimer and accordingly directed VDR-mediated transcriptional activation in a 1,25-(OH)2D3-dependent manner, as summarized in Fig. 6
. Importantly, 1,25-(OH)2D3 strongly induced Insig-2a expression in 3T3-L1 cells. Given the fact that SREBP-1c is a potent adipogenic factor (1, 2), our results suggest that the inhibitory action of 1,25-(OH)2D3 in adipogenesis (16, 17, 18, 19) may also involve up-regulation of antilipogenic protein Insig-2a (20, 21, 22), which will likely down-regulate the proadipogenic factor SREBP-1c. However, it is not known whether Insig-2 protein is actually increased in response to 1,25-(OH)2D3 because our studies measured only the level of mRNA. Additional studies are also warranted to directly determine whether increased Insig-2 expression upon 1,25-(OH)2D3 treatment actually results in decreased level of nuclear SREBP-1c in 3T3-L1 preadipocytes and blocks their adipogenesis. It should be noted that the strong induction of Insig-2a in 3T3-L1 cells was somewhat unexpected, given the recent report that described the lack of this isoform in brown and white fats (12). Along with the strict VDR dependence of this response (Fig. 3D
) and the suppression of VDR expression in fully differentiated adipocytes (Fig. 5
), it is possible that Insig-2a induction by 1,25-(OH)2D3 is only a transient event during adipogenesis and thus may not be observed in mature adipocytes, as reported (12). It is also an interesting possibility that this narrow window in which VDR and Insig-2a are transiently coexpressed (Fig. 5
) may represent a critical commitment switch controlled by 1,25-(OH)2D3/LCA, which senses whether it is safe to proceed with adipogenesis (i.e. lack of 1,25-(OH)2D3/LCA) or not (i.e. 1,25-(OH)2D3/LCA-directed antiadipogenesis). Further studies are warranted to test this exciting hypothesis.

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Fig. 6. Schematic Representation of the Working Model
The VDR/RXR heterodimer directly bound to Insig-2 VDRE induces Insig-2a expression upon binding 1,25-(OH)2D3 or secondary bile acids such as LCA derived from high cellular cholesterol level. Insig-2 in turn might shut off further generation of active SREBPs in adipocytes and other tissues/cell types in which VDR is expressed either constitutively or in response to certain signals.
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This novel regulatory circuit may also operate in other cell types and tissues, in which both VDR and Insig-2a are coexpressed (Fig. 6
). It is interesting to note that Insig-2a was recently described as a major hepatic Insig molecule in the presence of high cholesterol (12). The VDR-mediated regulation of Insig-2 observed in this report may thus represent one of the multiple hepatic safety mechanisms by which the liver senses the presence of too much cellular cholesterol (e.g. via a cholesterol metabolite LCA, a ligand for VDR). Because 1,25-(OH)2D3 itself is a metabolic product of cholesterol, this regulatory circuit could also be responsible for limiting further production of 1,25-(OH)2D3. However, as already noted, this possibility is severely hampered by the presence of only a limiting amount of VDR in hepatocytes (29), unless VDR is simultaneously induced by certain signals or metabolic cues. Consistent with the key regulatory role of Insig-2 in hepatic lipid homeostasis (12), however, we have found that Insig-2 promoter is subjected to multiple regulatory signaling pathways that are known to lower cellular lipid level, including metabolic nuclear receptors such as PPAR
and thyroid hormone receptor (our unpublished results). It is interesting to note that impaired insulin secretory capacity was recently observed in mice lacking VDR (32), and this phenotype could be linked to the lipotoxicity associated with overexpressed SREBP-1c in pancreatic ß-cells (33). These results suggest an intriguing possibility that the 1,25-(OH)2D3/Insig-2a regulatory circuit we described in this report may at least partially contribute to alleviating the lipotoxicity associated with increased SREBBP-1c activities (33). Indeed, we found that 1,25-(OH)2D3 induces the promoter activities of Insig2-fragment I-TK-Luc reporter in glucose-responsive ß-cell line INS-1 (34) (our unpublished results). Finally, we note that the Insig-2 promoter is a highly complex yet an excellent model system to study the metabolic regulatory network of a variety of transcriptional coregulatory proteins. In particular, our current emphasis has been focused on dissecting this promoter to further elucidate the role of a novel coactivator ASC-2 in lipid homeostasis (23).
In conclusion, we discovered a novel 1,25-(OH)2D3/LCA response element in the promoter region of Insig-2 that specifically binds the VDR/RXR heterodimer, and this element may direct VDR-mediated transcriptional stimulation of Insig-2 mRNA production in vivo. Our results suggest a novel VDR-mediated regulatory loop in lipid homeostasis, although further studies are warranted such as examining Insig-2 expression in primary adipocytes and other cell types from both wild type and VDR knockout mice (35).
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MATERIALS AND METHODS
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Plasmids and Ligands
PCR fragments containing Insig-2 fragments I, II, III, Ia, Ib, Ic, and Im were inserted into SalI and BamHI restriction sites of the TK-luciferase vector pTK-Luc. PCR fragment encoding VDR Y236A (27) was inserted into EcoRI and XhoI restriction sites of pcDNA3. Similarly, PCR fragments containing three copies of Insig-2 DR3 and Insig-2 VDRE were inserted into BamHI restriction site of pTK-Luc. Mammalian expression/T7 in vitro transcription vectors for VDR, FXR, LXR, RAR, PPAR
and RXR, the reporter constructs LXRE-Luc, pLTP-Luc, DR3-Luc, DR5-Luc and DR1-Luc, and the transfection indicator construct pRSV-ß-gal were as described previously (15, 23, 36). 1,25-(OH)2D3, LCA, CDCA, and 9-cis-RA were purchased from Sigma. T0901317 and rosiglitazone were gifts from Dr. Heonjoong Kang at Seoul National University, Korea.
Cell Culture and Transfection
3T3-L1 preadipocytes were grown in medium containing 10% (vol/vol) fetal bovine serum. Induction of differentiation was achieved by treatment of 2 d postconfluent cells with 1 µM Dex, MIX, and insulin (1.67 µg/ml), and insulin (1.67 µg/ml) alone from d 3. HepG2 or HEK293 cells were grown in 24-well plates with medium supplemented with 10% charcoal-stripped serum. After a 24-h incubation, cells were transfected with 100 ng of ß-galactosidase expression vector pRSV-ß-gal and 100 ng of Luc reporter gene along with expression vectors for indicated proteins using the Superfect Transfection Reagent (QIAGEN, Valencia, CA) according to the manufacturers protocol. Total amounts of expression vectors were kept constant by adding appropriate amounts of pcDNA3 to transfections. Three hours later, cells were washed and refed with DMEM containing 10% charcoal-stripped fetal bovine serum. After 24 h, cells were left unstimulated or stimulated with the indicated amount of each ligand. Cells were harvested 24 h later, luciferase activity was assayed as described previously (37), and the results were normalized to the ß-galactosidase expression. Consistent results were obtained in more than two similar experiments.
Gel Mobility Shift Assays
Gel mobility shift assays were performed as described previously (37). Radiolabeled Insig-2 VDRE oligonucleotides were incubated with in vitro-translated VDR and RXR, and the reaction products were analyzed by native polyacrylamide gel electrophoresis and autoradiography.
RT- and Q-PCR
Total RNA was isolated from Hepa1c1c7 or 3T3-L1 cells after lysis in TRIzol reagent according to the manufacturers protocol (Invitrogen Life Technologies, Carlsbad, CA), and RT-PCRs were performed as described previously (38). For the SYBR Green Q-PCR, 250 ng of cDNA was used per reaction. Each 25-µl SYBR Green reaction consisted of 5 µl of cDNA (50 ng/µl), 12.5 µl of 2x Universal SYBR Green PCR Master Mix (PE Biosystems, Foster City, CA), and 3.75 µl of 50 nM forward and reverse primers. Optimization was performed for each gene-specific primer before the experiment to confirm that 50 nM primer concentrations did not produce nonspecific primer-dimer amplification signal in no-template control tubes. Q-PCR was performed on ABI 5700 PCR Instrument (PerkinElmer Life Sciences, Boston, MA) by using three-stage program parameters provided by the manufacturer as follows: 2 min at 50 C, 10 min at 95 C, and then 40 cycles of 15 sec at 95 C, and 1 min at 60 C. Specificity of the produced amplification product was confirmed by examination of dissociation reaction plots. A distinct single peak indicated that single DNA sequence was amplified during PCR. In addition, end reaction products were visualized on ethidium bromide-stained 1.4% agarose gels, and appearance of a single band of the correct molecular size confirmed specificity of the PCR. Each sample was tested in triplicate with Q-PCR. Cyclophilin A mRNA levels were used as a reference standard. Primer sequences were designed using Primer Express Software (PerkinElmer Life Sciences) and are presented in Table 1
.
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ACKNOWLEDGMENTS
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We would like to thank Nancy Weigel, David D. Moore, and Heonjoong Kang for reagents and critical comments.
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FOOTNOTES
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This work was supported by grants from 21C Frontier Functional Proteomics Project FPR02A6-24-120 from the Korean Ministry of Science & Technology and National Institute of Diabetes and Digestive and Kidney Diseases R01-DK064678 (to J.W.L.).
First Published Online November 4, 2004
Abbreviations: Dex, Dexamethasone; DR, direct repeat; FXR, farnesol X receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Insig-1, insulin-induced gene-1; LCA, lithocholic acid; LXR, liver X receptors; MIX, 3-isobutyl-1-methylxanthine, 0.5 mM; 1,25-(OH)2D3, 1
,25-dihydroxyvitamin D3; PPAR, peroxisome proliferator-activated receptor; Q-PCR, quantitative PCR; RAR, retinoic acid receptor; RXR, retinoid X receptor; SCAP, SREBP cleavage activating protein; SREBP, sterol regulatory element-binding protein; TK, thymidine kinase; VDR, vitamin D receptor; VDRE, VDR response element.
Received for publication August 16, 2004.
Accepted for publication October 25, 2004.
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