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J. Biol. Chem., Vol. 281, Issue 30, 21096-21113, July 28, 2006

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Importance of cAMP-response Element-binding Protein in Regulation of Expression of the Murine Cyclic Nucleotide Phosphodiesterase 3B (Pde3b) Gene in Differentiating 3T3-L1 Preadipocytes^*

Hanguan Liu¹, Jing Rong Tang¹, Young Hun Choi, Maria Napolitano, Steven Hockman, Masato Taira, Eva Degerman², and Vincent C. Manganiello³

From the Pulmonary/Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and Section for Molecular Signaling, Department of Cell and Molecular Biology, University of Lund, S-22100 Lund, Sweden

Received for publication, February 10, 2006 , and in revised form, May 15, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Incubation of 3T3-L1 preadipocytes with isobutylmethylxanthine (IBMX), dexamethasone, and insulin, alone or in combination, demonstrated that IBMX, which increased cAMP-response element-binding protein (CREB) phosphorylation, was the predominant regulator of Pde3b expression. Real time PCR and immunoblotting indicated that in 3T3-L1 preadipocytes, IBMX-stimulated induction of Pde3b mRNA and protein was markedly inhibited by dominant-negative CREB proteins. By transfecting preadipocytes, differentiating preadipocytes, and HEK293A cells with luciferase reporter vectors containing different fragments of the 5'-flanking region of the Pde3b gene, we identified a distal promoter that contained canonical cis-acting cAMP-response elements (CRE) and a proximal, GC-rich promoter region, which contained atypical CRE. Mutation of the CRE sequences dramatically reduced distal promoter activity; H89 inhibited IBMX-stimulated CREB phosphorylation and proximal and distal promoter activities. Distal promoter activity was stimulated by IBMX and phorbol ester (PMA) in Raw264.7 monocytes, but only by IBMX in 3T3-L1 preadipocytes. Chromatin immunoprecipitation analyses with specific antibodies against CREB, phospho-CREB, and CBP/p300 (CREB-binding protein) showed that these proteins associated with both distal and proximal promoters and that interaction of phospho-CREB, the active form of CREB, with both Pde3b promoter regions was increased in IBMX-treated preadipocytes. These results indicate that CRE in distal and proximal promoter regions and activation of CREB proteins play a crucial role in transcriptional regulation of Pde3b expression during preadipocyte differentiation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cyclic 3',5'-nucleotide phosphodiesterases (PDEs)⁴ catalyze hydrolysis of cAMP and cGMP, important intracellular second messengers that regulate many signal transduction pathways. The PDE superfamily includes at least 11 distinct, structurally related, and highly regulated mammalian PDE gene families (PDE1-11) (1). Multiple PDEs are found in most cells but in different amounts, proportions, and subcellular locations; little is known of the mechanisms that regulate their differential expression. PDE3 isozymes can be distinguished from other PDE families by their high affinities for cAMP and cGMP, and by their sensitivity to specific inhibitors such as milrinone and cilostamide (1). The PDE3 family includes two isozymes, PDE3A and PDE3B, that have very similar pharmacological and catalytic properties and exhibit distinct, but overlapping, patterns of expression (2, 3). PDE3A is relatively highly expressed in the cardiovascular system, platelets, various types of smooth muscle, and oocytes (4); PDE3B is relatively highly expressed in cells critical to the maintenance of energy homeostasis such as white and brown adipocytes (4-7), hepatocytes (4, 8), and pancreatic -cells (9-11). PDE3B is also expressed in other tissues, including vascular smooth muscle (4, 12, 13).

cAMP seems to play a dual, if not seemingly paradoxical, role in adipocytes, and different PDEs may be involved in the regulation of these effects. cAMP is intimately involved in the differentiation program, presumably via activation of CREB, and also in promoting adipogenesis (14, 15). On the other hand, in mature or differentiated adipocytes, cAMP stimulates lipolysis via PKA-catalyzed phosphorylation of perilipin and phosphorylation/activation of hormone-sensitive triglyceride lipase (16). Expression of membrane-associated PDE3B is induced during differentiation of 3T3-L1 adipocytes (6, 17, 18). In differentiated adipocytes, insulin-induced phosphorylation and activation of membrane-associated PDE3B, most likely via phosphatidylinositol 3-kinase and protein kinase B, is an important component of the antilipolytic action of insulin (2, 19). Previous studies from our laboratories indicated that RO 20-1724 (a specific inhibitor of PDE4 isozymes), but not cilostamide (a specific PDE3 inhibitor), could replace IBMX (a non-selective PDE inhibitor) during differentiation of 3T3-L1 adipocytes induced by IBMX, dexamethasone, and insulin (20). We and others (21, 22) have also reported that specific inhibitors of PDE3, not PDE4, block the antilipolytic action of insulin in differentiated or mature adipocytes. Taken together, these observations suggest that PDE4 isoforms may regulate cAMP pools involved in initiation of differentiation, whereas PDE3 isoforms may regulate cAMP pools that control lipolysis in differentiated adipocytes. Experiments with PDE3 inhibitors have also indicated an important role for PDE3 in regulation of insulin secretion in pancreatic islets (9-11, 23, 24) and, perhaps, oxygen consumption in human subjects (25).

Because the detailed molecular mechanisms that govern adipocyte Pde3b expression during adipogenesis are not known, and because terminal differentiation of 3T3-L1 adipocytes provides a well characterized model for the study of gene expression and regulation (26, 27), we initiated studies of mechanisms underlying transcriptional control of Pde3b expression in these cells. We isolated the 5'-flanking region of the murine Pde3b gene, which included the putative first exon and an 5-kb genomic fragment upstream of the translation start site, and which contained distal and proximal promoter regions and transcription initiation sites in the proximal promoter region (0.5 kb upstream of the translation start site), close to those described by Niiya et al. (28). In addition, our results suggest that cis-acting CRE (cAMP-response) elements in both promoter regions and activation of CREB (CRE-binding proteins) are important in the regulation of induction of Pde3b by cAMP during differentiation of 3T3-L1 adipocytes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials
Tissue culture reagents (Dulbecco's modified Eagle's medium, fetal bovine serum, and penicillin/streptomycin), GeneRacer^TM kit, and TOPO TA Cloning® kit were from Invitrogen; [³H]cAMP was from PerkinElmer Life Sciences; rolipram, cilostamide, bisindolylmaleimide I, and anti-CREB antibodies were from Calbiochem; isobutylmethylxanthine, dexamethasone, and insulin were from Sigma; Affi-Gel 601 was from Bio-Rad; the BCA protein assay kit and bovine serum albumin were from Pierce. Enzymes for DNA manipulation were purchased from Roche Applied Science; [-³²P]dATP or [-³²P]ATP was from Amersham Biosciences. Vectors (basic-pGL3, SV40 promoter-pGL3, and SV40 promoter/enhancer-pGL3 which contained the firefly luciferase gene, and TK-pRL which contained the thymidine kinase (TK) promoter and the Renilla luciferase gene), TRANSFAST^TM reagent, the Dual Luciferase^TM reporter assay system, and gel shift binding buffer were obtained from Promega (Madison, WI). SMART^TM RACE cDNA amplification kits, AdvanTAge^TM PCR cloning kits, Advantage®-GC cDNA PCR kits, and Adeno-X kits were purchased from Clontech. Poly(A)⁺ Pure^TM mRNA isolation kits were from Ambion (Austin, TX); plasmid DNA purification kits and QuantiTECT CYBR GREEN kits were from Qiagen (Studio City, CA); QuickChange multisite-directed mutagenesis kits were from Stratagene (Cedar Creek, TX); BigDye® Terminator version 3.1 cycle sequencing kit was from Applied Biosystems (Foster City, CA); mouse 3T3-L1 preadipocytes, HEK293A cells, and RAW264.7 cells were from American Type Culture Collection (Manassas, VA).

Cell Culture
3T3-L1 preadipocytes and HEK293A cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a 95% air, 5% CO₂-humidified atmosphere. RAW264.7 cells were cultured under the same conditions but in RPMI1640 medium.

Treatment of Preadipocytes with Differentiation Agents
Preadipocytes were incubated for 5 days, or as otherwise indicated, in fresh media supplemented with isobutylmethylxanthine (IBMX, 300 µM), dexamethasone (Dex, 1 µM), and insulin (5 µg/ml), alone or in combination. Cells were harvested, homogenized on ice with buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM MgCl₂, 0.1 mM EGTA, 5 mM benzamidine, 1 µg/ml aprotinin, and 1 µg/ml leupeptin and centrifuged (1,000 x g, 5 min, 4 °C). Supernatant protein content was determined using the BCA protein assay (Pierce), with bovine serum albumin as standard.

cAMP Phosphodiesterase Assay
Portions of the supernatant fractions were assayed (30 min, 30 °C) in 100 µl containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl₂, 0.1 mM EGTA, and 1 µM [³H]cAMP (50,000 dpm). Assays were terminated with 50 µl of 0.5 M EDTA (pH 7.4). PDE3 activity was determined as that portion of total cAMP hydrolysis inhibited by cilostamide (a PDE3-selective inhibitor, 1 µM). After diluting samples with 0.3 ml of buffer (0.1 M NaCl, 0.1 M HEPES (pH 8.5)), the reaction product, 5'-[³H]AMP, was purified by polyacrylamide-boronate gel chromatography (Affi-Gel 601, 1-ml bed volume) and quantified by liquid scintillation counting.

Immunoblotting
Samples were mixed with 2x Laemmli buffer (125 mM Tris-HCl (pH 6.8), 50% glycerol, and 4% SDS), heated (95 °C, 5 min), and subjected to SDS-PAGE and Western blotting. Membranes were incubated (1 h) with blocking buffer (TBST (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20)) containing 5% powdered nonfat milk (NFDM) and then (1-2 h) with appropriate antibodies, washed, and incubated (1 h) with horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG. Immunoreactivity was detected by chemiluminescence. Recombinant mouse PDE3B was used to determine the specificity of the antiserum and for comparison of molecular masses.

Oil Red O Staining of Neutral Lipids
A stock solution of Oil Red O (5 mg/ml in isopropyl alcohol) was diluted to 3 mg/ml with distilled water, incubated (1 h, room temperature), and filtered (0.2-µm filter) prior to use (29). Cells were fixed with 10 ml of Accustain formalin solution (Sigma) in PBS (1 h, 4 °C) and stained with 10 ml of Oil Red O solution per dish (15 min, room temperature). Accumulation of neutral lipid (red color) was assessed using an Olympus IX51 microscope with a digital camera.

Adenovirus-mediated CREB Protein Expression in 3T3-L1 Preadipocytes
Recombinant adeno-CREB expression vectors, kindly supplied by J. Reusch and D. Klemm (Veterans Affairs Medical Center, Denver, CO), were constructed with replication-deficient adenovirus under control of the cytomegalovirus immediate early promoter that regulates the ectopic expression of cDNA encoding -galactosidase, inactive MCREB or KCREB, or constitutively active VP16-CREB or DCREB. Adenovirus constructs were amplified in HEK293A cells and purified with Adeno-X kits (Clontech). Adenovirus particles were quantitated by measuring absorbance at A_{260 nm}, and infection efficiency was determined by immunostaining with anti-adenovirus hexon protein or anti--galactosidase antibody. At 80% confluence, preadipocytes were infected for 3 h with adenovirus constructs (500 virus particles per cell) in DMEM containing 1 µg/ml polylysine. Cells were cultured in DMEM containing 2.5% FBS for 2 days, and after that, infected cells were incubated with or without 300 µM IBMX for additional 5 days in DMEM containing 10% FBS. Expression of Pde3b mRNA and protein was assessed by real time RT-PCR, immunoblotting, and PDE3 activity assays.

Poly(A)-mRNA Isolation from 3T3-L1 Preadipocytes and Differentiated Adipocytes
RNA was isolated from 3T3-L1 preadipocytes and differentiated adipocytes using poly(A) Pure^TM mRNA isolation kits (Ambion). After centrifugation (12,000 x g, 15 min, 4 °C) of cell extracts, supernatants were added to oligo(dT)-cellulose and incubated (1 h, room temperature with shaking). After washing the matrix, mRNA was eluted (100 µl of elution buffer, 10 min, 65-70 °C), pelleted with glycogen (12,000 x g, 20 min, 4 °C), and dissolved in diethyl pyrocarbonate-treated water.

Real Time RT-PCR Analysis
Real time RT-PCR was performed using Qiagen Quanti-TECT SYBR GREEN kit. Primers for amplification of mouse Pde3b, -actin, or cyclophilin A in cultured 3T3-L1 preadipocytes were Pde3b-3295 (5'-GGTGATGGTGGTGAAGAA-3') and Pde3b-3414 (5'-AGTGAGGTGGTGCATTAG-3'), actin-949 (5'-ATTACTGCTCTGGCTCCTA-3') and actin-1063 (5'-TCTGCTGGAAGGTGGACA-3'), and CypA-275 (5'-ATGTGGTCTTTGGGAAGG-3') and CypA-370 (5'-TGGTGATCTTCTTGCTGGT-3'), respectively. The relative abundance of the assayed gene (Pde3b) was calculated on the basis of Ct (threshold cycle) value (2^-Ct), Ct = the normalized signal level in a sample relative to the normalized signal level in the corresponding calibrator sample.

RNA Ligase-mediated Rapid Amplification of 5' cDNA Ends (RLM-5'-RACE) for Identification of Distal Transcripts of the Pde3b Gene
Poly(A)-mRNA was isolated from IBMX-treated 3T3-L1 preadipocytes using the Qiagen RNeasy mini kit and Oligotex mRNA mini kit. After first strand synthesis, cDNA was amplified by PCR using a GeneRacer 5'-primer and a gene-specific primer (5'-CAGCGGGCTCACGCAGCTCTTCACGTA-3', reverse-complement 100-126 bp downstream of ATG start codon). Nested PCR was performed on purified PCR products (Qiagen PCR purification kit), using a GeneRacer 5'-nested primer and a nested, gene-specific primer (5'-AGCTCTTCACGTAGCCGTTGCGCA-3', reverse-complement 89-112 bp downstream of ATG start codon). After TOPO TA cloning of PCR products, positive clones were identified by PCR and sequenced.

Transient Transfection with Transfast^TM Reagent (Promega) and the Dual Luciferase Assay
Cells were cotransfected with pRL-TK and pGL3 reporter plasmid vectors that expressed Renilla and firefly luciferase activities, respectively. For the different pGL3 reporter plasmids, firefly luciferase expression was driven either by Pde3b 5'-flanking region genomic fragments or constructs (Pde3b-pGL3), by the SV40 promoter (SV40 (pro)-pGL3) or the SV40 enhancer and promoter (SV40 (con)-pGL3) as positive controls, or by promoter-free pGL3 (Basic-pGL3) as a negative control.

Transfection was performed using TransFAST^TM reagent according to the manufacturer's instructions (Promega). On the day before transfection, 400 µl of nuclease-free water was added to the TransFAST^TM reagent, and the lipid film was dispersed by vigorous vortexing for 10 s. After 24 h, a mixture of plasmid DNA, TransFAST^TM reagent (3 µl), and serum-free growth medium (1 ml) was prepared, incubated (10-15 min, room temperature), and used to transfect cells, after removal of growth medium from the cells. Each DNA sample contained the pRL-TK plasmid vector (0.02 µg) and a pGL3 plasmid vector (1.0 µg). 3T3-L1 preadipocytes were cotransfected in 6-well culture dishes at 60-80% confluence, incubated for 1 h at 37 °C, and then overlaid with prewarmed DMEM containing 10% FBS (2 ml) and maintained for 48 h at 37 °C. For transfection of differentiating 3T3-L1 preadipocytes, preadipocytes were grown in DMEM containing 10% FBS to about 80% confluence (day 0), when differentiation was induced by addition of fresh DMEM containing 10% FBS and MDI (where MDI is IBMX, Dex, and insulin in differentiating preadipocytes). Three days later (day 3), differentiating 3T3-L1 preadipocytes were cotransfected by the procedure described above but were then overlaid with 2 ml of prewarmed DMEM containing 10% FBS and 1 µg/ml insulin and maintained for 48 h at 37 °C. After 48 h at 37 °C, the cotransfected cells were then washed three times with PBS, disrupted in 250 µl of Passive Lysis Buffer (PLB, Promega), and centrifuged (1,000 x g, 5 min). Supernatant firefly and Renilla luciferase activities were measured with the dual luciferase reporter assay system (Promega) using a luminometer (Lumat LB 9501). Luciferase reagent substrate (100 µl) was mixed with cell lysates (20 µl), and firefly luciferase activity was measured (10 s, twice). To terminate this reaction and initiate the Renilla luciferase reaction, Stop & GloR Solution (100 µl) was added to the mixture for 20 s, and Renilla luciferase activity was measured (10 s, twice). The firefly luciferase activity in lysates from cells transfected with Pde3b-pGL3 reporter constructs, and positive and negative controls, was normalized to Renilla luciferase activity values, and the ratio between firefly and Renilla luciferase activities was referred to as relative luciferase activity (RLA). RLA (mean ± S.D., n = 3) was assayed (in duplicate) in lysates from triplicate transfections as described above. Unless otherwise noted, experiments presented in the various figures are from single experiments, which were replicated once or twice.

For comparison of transcriptional activities in different cell types, HEK293A, 3T3-L1, and RAW264.7 cells were transfected with pGL3 plasmid DNAs containing various lengths of 5'-flanking regions of the Pde3b gene. After transfection, the cells were treated with IBMX (300 µM) for 3 days or PMA (50 nM) for 24 h and assayed for luciferase activities.

Construction of Pde3b-luciferase Vectors and Reporter Plasmids
In general, Pde3b reporter plasmids were generated by insertion of Pde3b genomic fragments and constructs into pGL3-Basic vectors.

Distal Region (-5.1 to -3.4 kb Upstream of ATG)—The 1.7-kb distal SalI/XbaI fragment from the 5'-flanking region of the Pde3b gene (-5.1 to -3.4 kb upstream of ATG) (Figs. 3A and 4) was ligated into SalI/XbaI sites of pBluescript, which were located between the KpnI and NheI restriction sites of pBluescript. The SalI/XbaI fragment (1.7 kb) was then excised from pBluescript by cleavage with KpnI/NheI and ligated into pGL3-Basic digested with KpnI/NheI, yielding SX-pGL3 (Figs. 4 and 5A). Direct ligation of the SalI/XbaI fragment into pGL3-Basic digested with NheI/XhoI yielded SX^*-pGL3, with the genomic fragment in the reverse orientation (Fig. 5A). The SalI/XbaI fragment in pBluescript was cleaved with KpnI/HindIII, KpnI/EcoRI, or KpnI/BamHI, and the fragments were ligated into pGL3-Basic vector at appropriate restriction sites to yield SH-pGL3 (350 bp), SE-pGL3 (1.1 kb), and SB-pGL3 (1.2 kb), respectively (Figs. 4 and 5A). The size and orientation of all constructs were verified by digestion with KpnI/BglII or KpnI/HindIII.

Proximal Region (3.4 kb Upstream of ATG)—To prepare constructs containing different portions of the 5'-flanking region of the Pde3b gene downstream of the distal SalI/XbaI fragment (Figs. 3A and 4), the 6-kb XbaI/XbaI fragment (Fig. 3A), which contained a BamHI restriction site just upstream of the initiation ATG codon (Fig. 3B), was subcloned, isolated, and digested with BfrII, NdeI, EcoRI, BsteII, PshAI, NotI, BglII, PvuI, or SmaI. The products with 5'- and 3'-overhang ends, together with the parent XbaI/XbaI fragment, were blunt-ended with Klenow enzyme and digested with BamHI to yield fragments with a 5'-blunt-end and a BamHI-restricted 3'-end (Figs. 3B and 4). These were subjected to electrophoresis (1% agarose gel), extracted (Qiagen, Quick Gel kit), and ligated to SmaI/BglII-digested pGL3-Basic (which contained a 5'-blunt end compatible with the 5'-blunt ends of the Pde3b genomic fragments and 3'-end compatible with BamHI) to form Xba-pGL3, Bfr-pGL3, Nde-pGL3, EcoR-pGL3, Bst-pGL3, Psh-pGL3, Not-pGL3, Bgl-pGL3, Pvu-pGL3, and Sma-pGL3 constructs (Figs. 3B and 4). A 0.8-kb ApaI/BamHI fragment from the XbaI genomic clone was ligated into pBluescript II SK(-), which was then digested with KpnI/BamHI. The excised fragment was then ligated into pGL3-Basic vector that had been treated with KpnI/BglII, generating Apa-pGL3. The sizes and orientation of the constructs (Fig. 4) were confirmed by digestion with KpnI/HindIII.

The entire 5'-upstream genomic SalI-BamHI fragment (5 kb) was ligated into pGL3-Basic vector (SXB-pGL3) via a three-fragment ligation, i.e. Sal-XbaI (1.7 kb) fragment excised from the MG171 genomic clone with KpnI-XbaI, the XbaI-BamHI fragment cut from the XbaI/XbaI genomic fragment, and pGL3-Basic digested with KpnI and BglII (Figs. 3B and 4). The size and orientation of constructs were verified by digestion with KpnI/EcoRI, ApaI/EcoRI, and other restriction enzymes.

Distal-Proximal Region Fusion Constructs—The 1.1-kb SalI-EcoRI portion (SE) from the SalI/XbaI distal genomic fragment (Figs. 4 and 5A), containing AP-2, Sp-1, and CRE cisacting elements, exhibited strong promoter activity when expressed as the SE-pGL3 reporter plasmid (Fig. 5A). SE was subcloned into SalI/EcoRI sites of pBluescript in between KpnI/SmaI sites, and then excised with KpnI/SmaI. Pde3b pGL3-Basic plasmids containing proximal promoter regions (Fig. 4) were digested with KpnI and MluI (restriction sites in the multicloning region of pGL3); the 5'-overhang resulting from MluI digestion was blunt-ended. The KpnI/SmaI fragment that contained the distal SE promoter region was ligated into the plasmids previously digested with KpnI/MluI (blunted) (Fig. 7). The resulting distal-proximal region fusion constructs contained a 13-bp (CGCGTGCTAGCCC) linker between the SE fragment of the distal promoter and the proximal promoter constructs (Fig. 7). One construct, SEEB, from which this linker was removed, was generated by digesting SE-Bfr-pGL3 with EcoRI to remove the Bfr-EcoRI portion of Bfr-pGL3, as well as the linker between distal and proximal promoter regions of the Pde3b gene, and by self-ligating at EcoRI sites (Fig. 7).

Construction of Expression Vectors Containing CRE, Sp-1, and AP-2 Sequences—A series of luciferase expression vectors, containing either the AP-2 (activator protein 2), Sp-1, or CRE sequences found in the SalI/XbaI fragment (Figs. 4 and 5C), were generated by PCR (forward and reverse primers are given in Table 1) from the SalI/XbaI fragment and named AT5-pGL3 (214 bp), AT4-pGL3 (125 bp), and AT1-pGL3 (224 bp), respectively (Fig. 5C). AT7-pGL3 (440 bp) contained AP-2, Sp-1 and CRE. A negative control luciferase expression vector containing a mutant CRE (TGACGTCAAAAAAAA) was named mAT1-pGL3 (224 bp). Other luciferase vectors, AT2-pGL3 (221 bp), AT3-pGL3 (317 bp), and AT6-pGL3 (460 bp), contained, respectively, combinations of AP-2/Sp-1, Sp-1/CRE, and AP-2/CRE. The genomic fragments in AT1-, mAT1-, AT2-, AT3-, AT4-, AT5-, and AT7-pGL3 were amplified with primer sets A859-T1058, A859-mT987, A660-T880, A748-T1064, A748-T861, A660-T851, and A660-T1064 (Table 1), respectively, under PCR conditions of 94 °C for 3 min followed by 30 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min followed by a 10-min extension at 72 °C. These fragments were subcloned into a TA-cloning vector, and the sequences were confirmed by automated sequencing with an ABI^TM PRISM dye terminator cycle sequencing ready reaction kit. Each fragment, recovered after digestion with SmaI/BglII, was ligated to a pGL3-Basic vector treated with SmaI/BglII. AT6-pGL3 was generated by ligation of AT5-pGL3 treated with BglII-blunted/HindIII and the insert from AT1-pGL3 digested with SmaI/HindIII. The sequence and orientation of the AT-pGL3 constructs were also confirmed by automated sequencing.

TGACGTCATGCGCGGG

Chromatin Immunoprecipitation Analysis
ChIP analyses were performed using an assay kit (Upstate%20Biotechnology">Upstate Biotechnology, Inc., Lake Placid, NY) according to the manufacturer's protocol. Confluent 3T3-L1 cells were cultured in DMEM containing 1% FBS and treated with or without 300 µM IBMX (16 h), after which cells were cross-linked with 1% formaldehyde (30 min, room temperature), washed twice with ice-cold PBS containing protease inhibitors, and harvested. Cell pellets were resuspended in SDS lysis buffer (10 min, 4 °C) and sonicated (Branson sonifier 450, power setting 5, duty cycle 50% for six times, each time for 25 s followed by 1 min on ice). After centrifugation, the supernatant was diluted in ChIP dilution buffer and incubated overnight at 4 °C with rabbit IgG, anti-CREB, anti-phospho-CREB, anti-ATF1, anti-CREM1, or anti-CBP and anti-p300 antibodies (Santa Cruz Biotechnology (Santa Cruz, CA)). Immune complexes were incubated (2 h, 4 °C, with rotation) with 60 µl of a 50% slurry of protein A-agarose beads containing salmon sperm DNA. Agarose beads were pelleted by gentle centrifugation (720 x g, 4 °C), sequentially washed with low and high salt buffer, LiCl buffer, and twice with TE buffer. After washing, the immune complexes were eluted by incubation (15 min, 25 °C) with 400 µl of 1% SDS, 0.1 M NaHCO₃. To reverse the cross-linking of DNA, 8 µl of 5 M NaCl was added, and the mixture was incubated (4 h, 65 °C). After treatment with proteinase K (1 h, 45 °C), DNA was recovered by phenol/chloroform extraction and ethanol precipitation and resuspended in 50 µl of TE buffer. Touch-down PCR (32 cycles, 3 µl of DNA) amplified the segment (-4326/-4092) flanking the consensus CRE site in distal promoter region with forward primer 5'-ATGGGTGGCCTATTTGCTCTTA-3' and reverse primer 5'-GGAGAATTCTCCTTCGCCTCG-3', and the segment (-698/-293) in the proximal promoter region containing putative CREB-binding sites with forward primer 5'-TGTGACTTACCTCAAAGGGGACT-3' and reverse primer 5'-CAGTTACCTCACGGCCGCAGCCT-3'. PCR products were separated on 2% agarose gels.

Statistical Analysis
Data are presented as means ± S.D. of at least three independent experiments. Within each experiment, values were means from three individual determinations for each experimental condition. Statistical analysis of ANOVA and Bonferroni-Dunn post hoc tests was performed to determine statistical differences among treatments with p < 0.05 considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Effects of Differentiation Agents on Pde3b Expression and Lipid Accumulation in 3T3-L1 Preadipocytes—As seen in Fig. 1A (lane 2), immunoreactive PDE3B protein was significantly increased during incubation of preadipocytes with IBMX alone (p < 0.01 compared with control). Dex and/or insulin, alone (Fig. 1A, lanes 3 and 4) or in combination (lane 7), only slightly increased Pde3b expression but significantly potentiated the effect of IBMX (lane 8) (p < 0.01). As shown in Fig. 1B, cilostamide-inhibited PDE activity, which in adipocytes represents mainly PDE3B activity (2-4, 6), was also increased in cells treated with IBMX, alone (lane 2) or in combination with Dex and/or insulin (lanes 5, 6, and 8). These results suggested that the PDE inhibitor IBMX, which increased (most likely via cAMP and PKA) phosphorylation of CREB in 3T3-L1 preadipocytes (cf. Fig. 8A), played a major role in the induction of Pde3b during differentiation and that insulin and dexamethasone (via unidentified signaling pathways) potentiated, or added to, the effects of cAMP. To assess effects of differentiation agents on neutral lipid accumulation, preadipocytes were incubated for 12 days in the presence of IBMX, Dex, and insulin, alone or in combination, and then stained with Oil Red O. As shown in Fig. 1C, in contrast to the effects on Pde3b expression, insulin, alone (lane 4) or in combination with IBMX and Dex (lanes 6-8), increased neutral lipid accumulation (quantitated by extraction of Oil Red O from the cells) to a much greater extent than IBMX (lane 2).

Inhibitory Effects of Dominant-negative CREB on Induction of Pde3b by IBMX—CREB is apparently a crucial transcriptional activator of mitotic clonal expansion and the differentiation program in adipocytes, because of its role in regulating transcription of C/EBP (14, 15, 30, 31). To examine further the role of CREB on Pde3b expression, without triggering differentiation, 3T3-L1 preadipocytes were infected with adenovirus constructs expressing -galactosidase (adeno--galactosidase), dominant-negative forms of CREB (MCREB and KCREB), or active forms of CREB (V16-CREB, D-CREB) and treated with 300 µM IBMX alone for 5 days. As seen in Fig. 2, IBMX increased expression of PDE3B immunoreactive protein (Fig. 2A) and PDE3 activity (Fig. 2B) in control preadipocytes (lane 3) and preadipocytes infected with adeno--galactosidase (lane 4), indicating that infection did not affect the induction process. IBMX-induced expression of Pde3b was enhanced by overexpression of active V-16 and D-CREB (Fig. 2B, lanes 5 and 8), and markedly decreased by dominant-negative forms of CREB, MCREB, or KCREB (lanes 6 and 7). As shown in Fig. 2C, IBMX (in preadipocytes) and MDI (IBMX, Dex, and insulin in differentiating preadipocytes) induced Pde3b gene transcription (quantified by real time PCR), which was inhibited by overexpression of KCREB. These data suggest that IBMX-induced expression of Pde3b in both preadipocytes and differentiating preadipocytes requires activation of CREB.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Effects of differentiation agents on PDE3 activity, induction of PDE3B protein, and accumulation of neutral lipids in differentiating 3T3-L1 adipocytes. Preadipocytes were incubated with IBMX (300 µM), Dex (1 µM), or insulin (5 µg/ml), alone or in combination, for 5 (A and B) or 12 (C) days. Cells were homogenized (20 mM Tris-HCl (pH 7.4), 1 mM MgCl2, 0.1 mM EGTA, 5 mM benzamidine, 1 µg/ml aprotinin, and 1 µg/ml leupeptin) and centrifuged (1,000 x g, 5 min, 4 °C). A, supernatants (30 µg of protein) were subjected to SDS-PAGE and immunoblotted with anti-PDE3B N-terminal peptide and anti--actin antibodies. After scanning densitometry (HP Scanjet 5550C flatbed scanner), the relative intensity of the immunoreactive bands was quantified with Adobe Photoshop version 5.0. The ratio of the relative intensity of PDE3B bands to those of -actin was calculated, and arbitrary units were assigned. B, PDE3 (cilostamide-inhibited) activity (pmol/min/mg protein) was assayed as described under "Experimental Procedures." C, neutral lipid accumulation was quantitated by the amount of Oil Red O extracted from the cells with isopropyl alcohol (absorbance at A540 nm). Results in A-C represent pooled data from three separate experiments (mean ± S.D.). Statistical analysis of ANOVA and Bonferroni-Dunn post hoc tests was performed with StatView (version 5.0.1). *, p < 0.05; **, p < 0.01.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Inhibition of IBMX-induced expression of Pde3b by ectopic dominant-negative CREB. Preadipocytes, infected with adenovirus constructs containing -galactosidase, MCREB, KCREB, V16-CREB, or DCREB, were incubated without or with 300 µM IBMX for 5 days. 100,000 x g membrane fractions were prepared. A, samples (20 µg of protein) were separated by SDS-PAGE and immunoblotted. Immunoreactive PDE3B bands were quantitated by densitometry scanning. A representative PDE3B blot from three experiments is shown. B, samples (5 µg of protein) were assayed for PDE3 (cilostamide-inhibited) activity (pmol/min/mg protein). C, 3T3-L1 preadipocytes were infected with either -galactosidase or KCREB adenovirus and treated for 4 days with IBMX (300 µM) alone or with MDI (IBMX (300 µM), Dex (1 µM), and insulin (5 µg/ml)). Real time RT-PCR (in triplicate) of Pde3b transcripts was performed as described under "Experimental Procedures." Transcripts of -actin and cyclophilin A were quantitated as endogenous controls. Results in A-C represent pooled data from three experiments (mean ± S.D.). Statistical analysis of ANOVA and Bonferroni-Dunn post hoc tests was performed with StatView (version 5.0.1). *, p < 0.05; **, p < 0.01.

View larger version (72K): [in this window] [in a new window]   FIGURE 3. Analysis of promoter regions in the 5'-flanking region of the murine Pde3b gene. A, top, the 5'-end of one SalI genomic clone (MG171, 14 kb) included SalI-XbaI (1. 7 kb) and XbaI-XbaI (6 kb) fragments, the latter of which contains the coding region of putative exon 1 (filled black bar). The two genomic fragments were subcloned into pBluescript and used to construct Pde3b-luciferase reporter vectors (Fig. 4). The 5'-flanking region of the Pde3b gene extends 5.1 kb upstream from the ATG translation initiation codon. Putative proximal and distal promoter regions are identified with arrows and depicted separately, with several putative transcription factor-binding sites. The ATG start codon is boldface; putative transcription initiation sites are marked. B and C, promoter activities of luciferase reporter gene constructs containing various fragments of the 5'-flanking region. Luciferase reporter expression vectors were constructed with the whole or indicated portions of the 5.1 kb SalI/BamHI fragment (Fig. 4). RLA (mean ± S.D., n = at least three separate experiments) was measured (duplicate assays) in lysates from triplicate transfections of 3T3-L1 adipocytes and differentiating preadipocytes (B) and of HEK293A cells incubated with or without IBMX (C), as described under "Experimental Procedures." RLA represents the fold increase in firefly luciferase activity normalized to Renilla luciferase activity. *, p < 0.05; **, p < 0.01, statistically different from control. Basic-pGL3 represents the promoter- and enhancer-free pGL3 vector.

The proposed locations of the distal and proximal promoter regions (upstream of the ATG translation start codon) are presented in Fig. 3A; their nucleotide sequences, which are homologous (99% identity) to the corresponding mouse genomic sequences deposited in GenBank^TM, are presented in Fig. S1, A and B. Alignment of GenBank^TM sequences indicated limited homology among the 5'-flanking regions of the mouse, rat, chicken, and human PDE3B gene; only the mouse contained the canonical CRE sequences in the distal promoter region. Atypical CREB-binding sites, however, were predicted in putative distal and proximal promoter regions in the 5'-flanking regions of the four species. Consistent with our experimental results, two promoter regions (beginning at position -4061 and another at -364) were predicted when 5105 bp of the 5'-flanking sequence of the murine Pde3b gene was queried online. On the other hand, analysis at another on-line site predicted one promoter (at -532).

Distal Promoter Activity—To characterize the distal promoter region, 3T3-L1 preadipocytes or HEK293A cells were transfected with Pde3b-pGL3 constructs generated from the 1.7 kb SalI/XbaI fragment in the upstream or distal promoter region (-5.1 kb to -3.4 kb) of the 5'-flanking region of the Pde3b gene (Figs. 3A, 4, and 5, A and B). As depicted in Fig. 5, A and B, SX-pGL3, SB-pGL3, and SE-pGL3, but not SH-pGL3, exhibited strong promoter activity, indicating that active ciselements were located between HindIII and BamHI sites in the SalI/XbaI fragment. The promoter activity of this region, which contains a canonical CRE site (TGACGTCA) and Sp-1 and AP-2 cis-acting elements, as well as a putative TATA box (Fig. 3A, Fig. 5A, and Fig. S1B), was much higher than that of the SV40 promoter-pGL3 construct (Fig. 5, A and B) and was increased in differentiating preadipocytes (Fig. 5A), as well as in 3T3-L1 preadipocytes and HEK293A cells (Fig. 5B) incubated with IBMX. As also seen in Fig. 5A, in preadipocytes, the activities of the distal promoter fragments (SX-pGL3, SB-pGL3, and SE-pGL3) were orders of magnitude greater than Basic-pGL3. This suggested that activities of these distal promoter fragments were also much greater than the promoter activities (Fig. 3B) of the entire full-length 5'-flanking promoter region (SXB-pGL3) or proximal promoter fragments (Apa-, PshA-, Not-, and Bgl-pGL3), because, in preadipocytes, the luciferase activities of the latter constructs were only severalfold greater than Basic-pGL3 (note the differences in scale in Figs. 3B and 5A). Distal promoter activity was orientation-dependent because the SX^*-pGL3 construct, with the same SalI-XbaI fragment as in SX-pGL3, but in reverse orientation, did not exhibit promoter activity (Fig. 5, A and B).

View larger version (36K): [in this window] [in a new window]   FIGURE 4. Size and position of the fragments from 5'-flanking region of the Pde3b gene in luciferase reporter expression vectors. The A of the translation start codon ATG is position +1.

Effects of PMA and IBMX on Promoter Activities and CREB Protein Phosphorylation in Raw264.7 Monocytes and 3T3-L1 Preadipocytes—As seen in Fig. 6, promoter activity of several distal region luciferase reporter vectors was stimulated by IBMX, but not phorbol ester (PMA) (50 nM), in transfected 3T3-L1 preadipocytes (Fig. 6A), whereas distal promoter activity was, under these conditions, stimulated by both IBMX and PMA in Raw264.7 monocytes (Fig. 6B). Mutation of CRE in the distal promoter SX mutCRE-pGL3 reporter vector significantly reduced the effect of PMA on luciferase reporter gene activities (Fig. 6C), suggesting that the CRE are important for the effects of PMA on Pde3b gene expression (Fig. 6B). In HEK293A cells transfected with SX-pGL3 (Fig. 6C) or with the NotI-pGL3 reporter vector (Fig. 6D) from the proximal promoter region (which contained an atypical CRE site), the effects of PMA on luciferase reporter gene activities were inhibited by bisindolylmaleimide I (a specific PKC inhibitor) and H89. The effects of IBMX were also blocked by H89 (Fig. 6D). PMA-induced CREB phosphorylation in monocytes (Fig. 6E) was more effectively inhibited by BisI than H89, suggesting that effects of PMA on CREB phosphorylation were mainly mediated by activation of PKC. (R_p)-cAMPS and H89 blocked the effects of IBMX. In HL-60 cells, PMA-induced expression of ganglioside GM3 synthase is thought to be mediated via PKC/mitogen-activated protein kinase-dependent CREB phosphorylation (33).

View larger version (43K): [in this window] [in a new window]   FIGURE 5. Promoter activities of constructs from the distal portion of the 5'-flanking region of the Pde3b gene. Luciferase reporter expression vectors were constructed with indicated portions of the distal 1.7-kb SalI/XbaI fragment, which contained the indicated restriction sites and AP-2, SP-1, and CRE cis-acting elements. RLA (mean ± S.D., n = 3 experiments) was measured (in duplicate) in lysates from triplicate transfections of 3T3-L1 preadipocytes and differentiating preadipocytes (A) and preadipocytes and HEK293A cells incubated without or with IBMX (B). SX*-pGL3 is orientation-reversed SX-pGL3 construct; mutCRE, mutTATA, and mutCRE/TATA are SX-pGL3 reporter vectors with mutations in CRE, TATA, and CRE and TATA sequences, respectively. C, HEK293A cells were transfected with luciferase reporter expression vectors that contained CRE, Sp-1, and AP-2 elements, alone and in combination, and that were generated by PCR from the 1.7-kb SalI/XbaI distal fragment, using primers listed in Table 1. AT1-pGL3 and mAT1-pGL3 reporter vectors contained wild-type and mutant CRE sequences, respectively. Transfected cells were incubated with IBMX. RLA (mean ± S.D., n = at least three separate experiments) was measured (duplicate assays) in lysates from triplicate transfections. A-C, SV40 (Pro)-pGL3 refers to pGL3 expression vector driven by the SV40 promoter; SV40(Con)-pGL3, pGL3 expression vector driven by both the SV40 promoter and SV40 enhancer; Basic-pGL3, the pGL3-promoter- and enhancer-free vector. **, p < 0.01, statistically different from control.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Effects of PMA or IBMX on distal and proximal promoter activities. 3T3-L1 preadipocytes (A) and Raw264.7 monocytes/macrophages (B) were transfected for 6 h with luciferase reporter expression vectors containing portions from the distal promoter 1.7-kb SalI/XbaI fragment or orientation-reversed Sal/XbaI (SX*) fragment. Cells were incubated with either IBMX (300 µM) for 3 days or PMA (50 nM) for 24 h and then lysed, and RLA was assayed. C and D, HEK293A cells were transfected for 6 h with luciferase reporter expression vectors containing either the 1.7-kb Sal/XbaI reporter vector (SX-pGL3) with or without mutations at the CRE site (C) or the NotI/BamHI fragment which contains the proximal promoter region (D). C and D, cells were incubated for 24 h with either PMA (50 nM) alone, PMA + BisI (2.5 µM), PMA + H89 (10 µM) (C) or PMA (50 nM) alone, PMA + BisI (2.5 µM), PMA + H89 (10 µM), IBMX (300 µM), IBMX + H89 (10 µM) (D) and then lysed, and RLA was assayed (triplicate assays). Data are presented as mean ± S.D. (n = 3 experiments). *, p < 0.05; **, p < 0.01, comparison of treated to control or comparison between treated with or without inhibitors. E, effects of inhibitors on PMA- and IBMX-induced CREB phosphorylation in Raw264.7 monocytes. Raw264.7 monocytes/macrophages were incubated with DMEM for 16 h and then with H89 (10 µM), (Rp)-cAMPS (10 µM), or BisI (2.5 µM) for 20 min, and then for 2 h with IBMX (300 µM) or PMA (50 nM), alone or in combination. Cells were lysed in protein sample buffer containing 2% SDS, 10 mM -glycerol phosphate, 5 mM benzamidine, 50 mM sodium fluoride, and 1 mM sodium orthovanadate. Samples (30 µg of protein) were subjected to SDS-PAGE and immunoblotted with anti-phospho-CREB or -CREB1 antibodies; relative immunoreactive band intensities were quantitated by densitometry scanning. Data, mean ± S.D. (n = 3 experiments), were statistically analyzed with ANOVA, and Bonferroni-Dunn post hoc tests were performed with StatView (version 5.0.1). *, p < 0.05; **, p < 0.01. A representative blot from three separate experiments is shown. Ctrl, control.

Because the results in Figs. 5, 6, 7, 8 demonstrated the importance of CRE cis-acting elements in the regulation of Pde3b expression, and because the distal promoter region contained a canonical CRE consensus sequence at -4166 to -4159 and a putative TATA box at -4093 to -4084 (Fig. S1B), we speculated that Pde3b gene transcription might be driven by the distal promoter. Thus, as described in the Supplemental Material (cf. Fig. S1B), we attempted to use RT-PCR, RLM-5'-RACE, and RPA to identify transcripts that might be generated from the distal promoter region. However, multiple primer sets failed to amplify any Pde3b transcripts in the distal promoter region (data not shown). Also, RPA did not detect any protected RNA fragments following hybridization of adipocyte mRNA with antisense RNA probes designed based on sequence (nucleotides -4150 to -3867) from the distal promoter region of the Pde3b gene (data not shown). Although endogenous transcripts generated from the distal promoter region of the Pde3b gene were not identified, as seen in supplemental Fig. S1C, a fusion transcript generated in cells transfected with SX-pGL3 reporter vector (Fig. 4) indicated that there was transcription starting from SX and fusing to the firefly luciferase gene, which implied that the distal promoter could initiate transcription of an unidentified, noncoding exon or alternative exon1 from the murine Pde3b gene.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
PDE3B appears or is markedly increased during differentiation of 3T3-L1 adipocytes in the presence of MDI (6, 17, 18). In adipocytes, PDE3B regulates cAMP pools important for lipolysis and the antilipolytic action of insulin but not for initiation of differentiation (20-22). In addition, the presence of fat depots in PDE3B-deficient mice, recently generated in our laboratory (data not shown), also indicates that PDE3B is not required for differentiation. These data, however, do not rule out an important role(s) for Pde3b expression during differentiation or its impact on adipogenesis.

During differentiation in the presence of MDI, IBMX, which increased CREB phosphorylation, was the predominant regulator of Pde3b expression (Figs. 1 and 2). ChIP analysis indicated that phosphorylated CREB interacts with CRE cis-acting elements in the proximal and distal promoter regions of the 5'-flanking region of the Pde3b gene and that phosphorylated CREB interacts with and thereby recruits the multifunctional coactivator, CBP/p300 histone acetyltransferase, to Pde3b promoter regions. In keeping with the current hypotheses concerning coactivators and histone acetyltransferases (35, 36), CBP/p300, by acetylating nucleosomal histone tails, could make the Pde3b promoter regions more accessible to other regulatory factors. CBP/p300 might also function as a scaffold for other transcription factors/coactivators or a bridge between Pde3b-specific factors and the general transcription factors and RNA polymerase II, and thus initiate transcription of Pde3b. In addition, CBP/p300 can catalyze covalent acetylation of proteins, including transcription factors and coactivators. Although CBP/p300 most likely is a critical coactivator for CREB-mediated regulation of Pde3b transcription, the relative importance of histone acetyltransferase activities, scaffold functions, and protein acetyltransferase activities in regulation of assembly of the transcriptional complex and Pde3b gene expression is not known.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Promoter activities of proximal/distal promoter fusion reporter vectors expressed in preadipocytes and differentiating preadipocytes. The SE fragment from the distal SalI/XbaI region (Figs. 4 and 5A) was coupled to the indicated fragments from the proximal promoter region (Figs. 3B and 4). Restriction sites used to generate the constructs are indicated. Promoter activity of the transfected proximal/distal promoter fusion constructs was measured as RLA (mean ± S.D., n = 3 experiments) as described under "Experimental Procedures." , the 13-bp linker present between the SE fragment and the proximal promoter fragments. *, p < 0.05.