Originally published In Press as doi:10.1074/jbc.M314166200 on March 15, 2004
J. Biol. Chem., Vol. 279, Issue 21, 21740-21748, May 21, 2004
Small Interfering RNA Knockdown of Calcium-independent Phospholipases A2 
or 
Inhibits the Hormone-induced Differentiation of 3T3-L1 Preadipocytes*
Xiong Su
,
David J. Mancuso
,
Perry E. Bickel¶,
Christopher M. Jenkins, and
Richard W. Gross||**
From the
Division of Bioorganic Chemistry and Molecular Pharmacology, Departments of Internal Medicine, Chemistry, ||Molecular Biology & Pharmacology, and ¶Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, December 24, 2003
, and in revised form, February 18, 2004.
 |
ABSTRACT
|
|---|
Alterations in lipid secondary messenger generation and lipid metabolic flux are essential in promoting the differentiation of adipocytes. To determine whether specific subtypes of intracellular phospholipases A2 (PLA2s) facilitate hormone-induced differentiation of 3T3-L1 cells into adipocytes, we examined alterations in the mRNA level, protein mass, and activity of three previously characterized mammalian intracellular PLA2s. Hormone-induced differentiation of 3T3-L1 cells resulted in 7.3 ± 0.5- and 7.4 ± 1.4-fold increases of mRNA encoding the calcium-independent phospholipases, iPLA2
and iPLA2
, respectively. In contrast, the temporally coordinated loss of at least 90% of cPLA2
mRNA was manifest. Western analysis demonstrated the near absence of both iPLA2 and iPLA2 protein mass in resting 3T3-L1 cells that increased dramatically during differentiation. In vitro measurement of PLA2 activities demonstrated an increase in both iPLA2 and iPLA2 activities that were discriminated using the chiral mechanism based inhibitors (S)- and (R)-BEL, respectively. Remarkably, treatment of 3T3-L1 cells with small interfering RNA directed against either iPLA2 or iPLA2 prevented hormone-induced differentiation. Moreover, analysis of the temporally programmed expression of transcription factors demonstrated that the small interfering RNA knockdown of iPLA2 or iPLA2 resulted in down-regulation of the expression of peroxisome proliferator-activated receptor and the CCAAT enhancer-binding protein (C/EBP). No alterations in the expression of the early stage transcription factors C/EBP and C/EBP
were observed. Collectively, these results demonstrate prominent alterations in intracellular PLA2s during 3T3-L1 cell differentiation into adipocytes and identify the requirement of iPLA2 and iPLA2 for the adipogenic program that drives resting 3T3-L1 cells into adipocytes after hormone stimulation.
|
INTRODUCTION
|
|---|
Recently, there has been a dramatic increase in the incidence of obesity in industrialized and newly developed countries (1). Obesity results from abnormal increases in white adipose tissue (WAT)1 mass leading to alterations in whole organism energy storage and utilization (2–4). Increased adipose tissue mass can result from either an increase in individual adipocyte cell size (hypertrophy) or from an increase in total adipocyte number (hyperplasia). Alterations in whole organism lipid homeostasis leading to increased adipocyte tissue mass are highly correlated with the metabolic syndrome that is accompanied by its lethal sequelae of diabetes, hypertension, and atherosclerosis (2–5). During the last decade, substantial progress has been made in understanding the biochemical events leading to adipocyte differentiation utilizing the hormone-induced 3T3-L1 cell model of adipocyte differentiation (6–9). Central to this understanding has been the detailed characterization of temporally coordinated changes in the expression of specific genes that collectively define the adipocyte phenotype. Differentiation of adipocytes is accomplished by the programmed activation of transcriptional regulatory proteins that modulate the expression of mRNA and proteins that effectively reprogram 3T3-L1 cell lipid metabolism to that of a mature adipocyte. Such alterations include increased de novo fatty acid synthesis, accumulation of perilipin-coated triglyceride droplets, and the generation of lipid secondary messengers including eicosanoids and lysophosphatic acid that serve as potent and specific regulators of coordinated differentiation programs (3, 7, 10–14).
Phospholipases A2 (PLA2s) catalyze the hydrolysis of the sn-2 fatty acid substituents from glycerophospholipid substrates to yield free fatty acid (e.g. arachidonic acid) and lysophospholipid (15–17). Mammalian phospholipases A2 have been categorized into several classes based on their requirement for calcium ion in in vitro activity assays (i.e. millimolar, nanomolar, or no calcium requirement) leading to their broad classification into three classes of enzymes: calcium-independent phospholipase A2 (iPLA2), cytosolic phospholipase A2 (cPLA2), and secretory phospholipase A2 (sPLA2) (18). Prior studies have demonstrated that eicosanoids are potent modulators of adipocyte differentiation underscoring the roles of PGE2 and PGI2 in inducing transformation of progenitor cells into mature adipocytes (19, 20). In contrast, PGF2 inhibits hormone-induced differentiation of 3T3-L1 cells into mature adipocytes (21). In most mammalian cells, the rate-determining step in the production of biologically active eicosanoids is the release of arachidonic acid from the sn-2 position of glycerophospholipids. Despite the known importance of eicosanoids in modulating adipocyte differentiation, there is a paucity of information on the molecular identity of the specific types of intracellular phospholipases A2 present in differentiating adipocytes, the alterations in protein mass and activity levels of the different intracellular phospholipase A2 classes, and the importance of each specific type of phospholipase A2 in adipocyte differentiation (14).
Recent studies have demonstrated that lysophosphatidic acid (LPA) serves a dual function in adipocyte differentiation acting both as an extracellular ligand for EDG receptors (22, 23) and as the endogeneous intracellular ligand for the adipocyte transcriptional regulator peroxisome proliferator-activated receptor (PPAR) (24). According to current dogma, LPA produced during adipocyte differentiation results from the sequential hydrolysis of phosphatidylcholine to lysophosphatidylcholine (LPC) by endogenous phospholipases A2 and the subsequent extracellular hydrolysis of LPC to LPA catalyzed by a secreted lysophospholipase D, autotaxin (22). However, there is no information presently available on the types of phospholipases A2 present in the adipocyte that contribute to eicosanoid and lysolipid production in the adipocyte.
Recent analyses of the transcriptional programs utilized for adipocyte differentiation have identified the critical roles of the CCAAT/enhancer-binding protein (C/EBP) family and PPAR in mediating the transcriptional alterations required for adipocyte differentiation (3, 10). Hormone-induced growth-arrested 3T3-L1 cells treated with insulin, methylisobutylxanthine, and dexamethasone express the early transcription factors C/EBP and C/EBP, which lead to their re-entry into the cell cycle (25, 26). C/EBP and C/EBP then activate the transcription of C/EBP and PPAR, which are believed to both be antimitotic and act synergistically to activate the expression of adipocyte-specific genes leading to the differentiated adipocyte phenotype (27, 28).
In this study, we demonstrate the dramatic up-regulation of both iPLA2 and iPLA2 mRNA levels, protein content, and enzymatic activities during hormone-induced differentiation of 3T3-L1 cells temporally coordinated with the down-regulation of cPLA2 to near background levels. Moreover, the essential roles of iPLA2 and iPLA2 in adipocyte differentiation and their interplay with C/EBP and PPAR transcription factors have been identified by specific siRNA knockdown of either iPLA2 or iPLA2 activity. The results demonstrate that downregulation of iPLA2 or iPLA2 inhibits adipocyte differentiation via preventing PPAR and C/EBP expression without affecting the expression of C/EBP and C/EBP. Collectively, these results are the first to demonstrate the central roles of both iPLA2 and iPLA2 in the differentiation of a mammalian preadipocyte cell line into adipocytes.
|
EXPERIMENTAL PROCEDURES
|
|---|
Materials—3T3-L1 cells were obtained from ATCC (Manassas, VA). Fetal calf serum and Dulbecco's modified Eagle's medium (DMEM) were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum was obtained from BioWhittaker, Inc. (Walkersville, MD). Reagents for reverse transcription and quantitative PCR were supplied from Applied Biosystems (Foster City, CA). Oligonucleotide primer pairs and probes used in quantitative PCR were ordered from Applied Biosystems (Foster City, CA). SiRNA construction and transfection kits were purchased from Ambion (Austin, TA). All radiolabeled lipids were obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO). Most other chemicals were obtained from Sigma. Anti-PPAR, anti-C/EBP, anti-C/EBP, anti-C/EBP, anti-SCD I, and anti-cPLA2 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiperilipin and anti-GLUT4 antibodies were kindly provided by Dr. Michael M. Mueckler (Washington University, St. Louis, MO). Anti-PMP70 antibody was obtained from Affinity Bioreagents (Golden, CO). Rabbit anti-iPLA2 or anti-iPLA2 polyclonal antibodies were produced utilizing the synthetic peptides CEFLKREFGEHTKMTDVKKP (iPLA2) or CENIPLDESRNEKLDQ (iPLA2) and immunoaffinity purified as previously described (29).
Cell Culture of 3T3-L1 Cells and Differentiation into the Adipoctye Phenotype—3T3-L1 cells were cultured to confluence in DMEM containing 10% calf serum by changing the medium every 2 days as previously described (30). Two days after cell confluence, differentiation was initiated by adding differentiation medium 1 (0.5 mM methylisobutylxanthine, 0.25 µM dexamethasone, 1 µg/ml insulin in DMEM containing 10% fetal bovine serum). Two days later, methylisobutylxanthine and dexamethasone were removed and insulin (1 µg/ml) was maintained for 2 more days. Thereafter, cells were grown in DMEM containing 10% fetal bovine serum in the absence of differentiating reagents by replacing the media every 2 days.
Reverse Transcription and Quantitative PCR—Total RNA was purified from 3T3-L1 cell pellets utilizing a RNeasy® Mini Kit from Qiagen (Valencia, CA) according to the manufacturer's instructions. For cDNA preparation, 250 pmol of random hexamers were hybridized by incubation for 10 min at 25 °C and extended by incubation for 30 min at 48 °C in the presence of 125 units of reverse transcriptase in 100 µl of PCR buffer (5.5 mM MgCl2, 0.5 mM of each dNTP, and 40 units of RNase inhibitor). Reverse transcriptase was inactivated by incubation at 95 °C for 5 min. Amplification of each target cDNA was performed with TaqMan® PCR reagent kits and quantified by the ABI PRISM 7700 detection system according to the protocol provided by the manufacturer (Applied Biosystems, Foster City, CA). A traditionally utilized standard gene, glyceraldehyde-3-phosphate dehydrogenase, was measured and used as internal standard. Oligonucleotide primer pairs and probes specific for cPLA2 (5'-CCTTTGAGTTCATTTTGGATCCTAA/5'-TGTAGCTGTGCCTAGGGTTTCAT/5'-AGGAAAATGTTTTGGAGATCACACTGATGGATG), iPLA2 (5'-CCTTCCATTACGCTGTGCAA/5'-GAGTCAGCCCTTGGTTGTT/5'-CCAGGTGCTACAGCTCCTAGGAAAGAATGC), and iPLA2 (5'-GAGGAGAAAAAGCGTGTGCTACTTC/5'-GGTTGTTCTTCTTAAGGCCTGAA/5'-TCTGTTATCAATACTCACTCTTGCAATA) were employed.
Protein Extraction and Western Blot—Proteins from 3T3-L1 cells were extracted as described previously (31). Briefly, the cell monolayer was washed with ice-cold PBS and subsequently scraped into 1 ml of ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM phenylmethylmethanesulfonyl fluoride, 2 µg/ml aprotinin, and 1 µg/ml leupeptin). The solution was incubated on ice for 10 min after vortexing for 10 s. The cell homogenate was spun at 10,000 x g at 4 °C in a tabletop centrifuge for 10 min and the supernatant was transferred to a new tube and stored at –70 °C until used for Western blot analysis. Nuclear extracts were prepared with NE-PER® Nuclear and Cytoplasmic Extraction Reagents from Pierce according to manufacturer's protocol. Proteins were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore) in 10 mM CAPS buffer (pH 11) containing 10% methanol. Powdered milk (5% (w/v)) was used to block nonspecific binding sites prior to incubation with primary antibody directed against each specific protein as indicated. After incubation with secondary antibody (IgG-HRP conjugate diluted 1:5000 in blocking buffer), proteins were visualized by enhanced chemiluminescence according to the instructions of the manufacturer (Amersham Biosciences).
Phospholipase A2 Assays—On the day of the experiment, 3T3-L1 cells at different stages of differentiation were washed briefly with PBS and detached by incubation in trypsin-EDTA (0.25%, w/v) at 37 °C for 5 min. The cells were washed again with 5 volumes of CMRL-1066, transferred to a 50-ml Falcon centrifuge tube, and centrifuged for 5 min at 1700 rpm at 4 °C. The resulting cell pellets were resuspended in CMRL-1066 medium and centrifuged as above two more times. The cell pellets from 4 plates (10 mm diameter) were resuspended in 3 ml of lysis buffer (0.25 M sucrose, 25 mM imidazole, pH 7.2) and were sonicated six times for 1 s each. The tubes were placed on ice for 3 min and then re-sonicated. PLA2 assays were performed as described previously (32). Briefly, PLA2 activity were assessed by incubating 3T3-L1 cell protein (100–200 µg) with radiolabeled phosphatidylcholine, L--[oleoyl-1-14C]palmitoyl-2-oleoyl (POPC, 50 mCi/mmol, 5 µM final concentration, introduced by ethanol injection (2 µl)) in assay buffer (final conditions: 100 mM Tris-HCl, 4 mM EGTA, pH 7.2) at 37 °C for 30 min in a final volume of 200 µl. Reactions were quenched by addition of butanol (100 µl). 30 µlofthe organic phase of each sample were spotted on a Whatman silica plate that was developed with a nonpolar acidic mobile phase (100 ml of 70/30/1, petroleum ether/ethyl ether/acetic acid). Spots corresponding to fatty acids were scrapped into scintillation vials and radioactivity was quantified by scintillation spectrometry as described previously (32). BEL enantiomers were resolved by chiral high performance liquid chromatography as described previously (32). For the inhibition assays of iPLA2 by BEL, proteins were incubated with 10 µM (R)-BEL, (S)-BEL, racemic BEL, or ethanol vehicle for 3 min at 22 °C prior to the addition of radiolabeled substrate.
SiRNA Construction and Transfection—The siRNAs directed against iPLA2 and iPLA2 were constructed employing the SilencerTM siRNA construction kit (Ambion) according to the protocol provided by manufacturer. Upon confluence, the 3T3-L1 cell media were changed to growth media without antibiotics. One to 2 days later, cells were transfected with siRNAs (20 nM) using the siPORTTM lipid transfection reagent (Ambion) according to the manufacturer's instructions. Five volumes of 1.2x differentiation medium 1 without antibiotics were added 4 h after transfection and the cells were maintained at normal growing conditions and induced to differentiate as described above. Among four siRNAs for each targeting gene, the sequences specific for iPLA2 (5'-AACAGCACAGAGAAUGAGGAG-3') and iPLA2 (5'-AAGAUAAACAGCUUCAGGACA-3') were selected based upon their potency to inhibit target gene expression. A scrambled siRNA was used as a negative control.
Triacylglycerol Extraction and Electrospray Ionization Mass Spectrometry—After siRNA transfection, 3T3-L1 cells were grown to day 8 as described above. The cell monolayer was washed with ice-cold PBS and scraped into 1 ml of 50 mM LiCl. The lipids were extracted by the method of Bligh-Dyer (33) in the presence of an internal standard (Tri17:0TAG, 200 nmol/mg of protein). Mass spectral analysis of TAG was performed by electrospray ionization utilizing a Finnigan TSQ Quantum spectrometer (Finnigan MAT, San Jose, CA) as previously described (34).
Protein Extraction from White Adipose Tissue of Zucker Rats—Female obese Zucker (fa/fa) rats and lean congenic controls (5–6 weeks old) were housed and maintained with a 12-h light/12-h dark photoperiod. Water and food were given ad libitum. Animals were sacrificed (asphyxiated by CO2) and inguinal fat pads (WAT) were removed, rapidly frozen in liquid nitrogen, and ground with a motor and pestle. To the tissue powder was added lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM phenylmethylmethanesulfonyl fluoride, 2 µg/ml aprotinin, and 1 µg/ml leupeptin) and the resulting mixtures were homogenized with a Potter-Elvehjem apparatus. The homogenates were spun at 10,000 x g at 4 °C in a tabletop centrifuge for 10 min and the supernatant was transferred to a new tube and stored at –70 °C until used for Western blot analysis.
Miscellaneous—Protein concentration was determined utilizing a BCA protein assay kit (Pierce) with bovine serum albumin as a standard. All data were normalized to protein content and are presented as the mean ± S.E. Statistically significant differences between mean values were determined using unpaired Student's t tests.
|
RESULTS
|
|---|
Alterations in the mRNA Levels of Intracellular Phospholipases A2 during Differentiation of 3T3-L1 Preadipocytes— Prior work has underscored the essential roles of eicosanoid metabolites and LPC-derived LPA in adipocyte differentiation (19–22). Because these metabolites are all downstream products of PLA2 catalyzed reactions, we sought to determine the specific types and amounts of PLA2 mRNA, protein, and activity corresponding to each of the previously characterized mammalian intracellular PLA2 as a function of time after hormone-induced differentiation of 3T3-L1 preadipocytes. In resting cells, cPLA2 mRNA was prominent, with only minimal amounts of mRNA encoding iPLA2 detectable. However, after hormone-induced differentiation, the levels of cPLA2 mRNA decreased dramatically to near background levels (Fig. 1A). Remarkably, the levels of iPLA2 and iPLA2 mRNA increased 7.3 ± 0.5- and 7.4 ± 1.4-fold respectively (Fig. 1, B and C). Collectively, these results demonstrate the dramatic and temporally coordinated changes in the mRNA levels of each of the previously characterized mammalian intracellular PLA2 during adipocyte differentiation.
Alterations of Intracellular Phospholipase A2 Protein Mass and Activity during Differentiation of 3T3-L1 Preadipocytes—To further substantiate the functional importance of the observed alterations in mRNA levels, Western blot analysis was performed. Western analyses demonstrated a decrease in cPLA2 protein mass to near background levels (as predicted by the decreased mass content of cPLA2 mRNA in the differentiating adipocyte) and the dramatic increases of both iPLA2 and iPLA2 protein products (as predicted by increased mRNA levels encoding iPLA2 and iPLA2 from quantitative PCR) (Fig. 2). The temporal course of the increased amounts of iPLA2 and iPLA2 protein and the decreased amount of cPLA2 protein were inversely regulated. Thus, the protein mass of each intracellular PLA2 closely paralleled the intrinsic mRNA levels of each of three mammalian intracellular PLA2 (i.e. cPLA2, iPLA2, and iPLA2). Collectively, these results demonstrate the importance of transcriptional regulation in modulating reciprocal alterations in specific classes of intracellular PLA2 during adipocyte differentiation.
To further investigate if alterations in the protein content of iPLA2 and iPLA2 present during differentiation of 3T3-L1 cells were paralleled by changes in their activities, phospholipase A2 activity assays were performed. During adipocyte differentiation iPLA2 activity increased 
4-fold (Fig. 3A). As anticipated, the measured increase in iPLA2 activity was inhibited by the mechanism-based inhibitor, racemic BEL (Fig. 3B). Previously, we demonstrated that (S)-BEL was approximately 1 order of magnitude more selective for iPLA2 in comparison to iPLA2, whereas (R)-BEL was approximately an order of magnitude more selective for iPLA2 (32). The measured iPLA2 activity in 3T3-L1 adipocyte homogenate was inhibited to similar levels by either (S)-BEL or (R)-BEL (Fig. 3B) demonstrating that both iPLA2 and iPLA2 contribute similarly to the total amounts of measured iPLA2 activity in differentiated adipocytes. Concomitant with the increase in iPLA2 activity, calcium-dependent PLA2 activity in the homogenate decreased by 90% in day 8 3T3-L1 cells (data not shown). Collectively, these results showed an increase of iPLA2 activity and a concomitant decrease of calcium-dependent phospholipase A2 activity.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 3.
In vitro calcium independent phospholipase A2 activities in 3T3-L1 cells during differentiation and their inhibition by BEL. 3T3-L1 cells were cultured, induced to differentiate, and at the indicated differentiation stages, cell homogenates were prepared as described under "Experimental Procedures." Phospholipase A2 activity was assessed by incubating 3T3-L1 cell protein (100–200 µg) with radiolabeled L--[oleoyl-1-14C]palmitoyl-2-oleoyl (POPC, 50 mCi/mmol, 5 µM final concentration, introduced by ethanol injection (2 µl)) in assay buffer (final conditions: 100 mM Tris-HCl, 4 mM EGTA, pH 7.2) at 37 °C for 30 min in a final volume of 200 µl. Reactions were quenched by addition of butanol (100 µl) and lipids were separated by TLC as described under "Experimental Procedures." Spots corresponding to fatty acids were scrapped into scintillation vials and radioactivity was quantified by scintillation spectrometry. A, iPLA2 activities of 3T3-L1 cells during differentiation. B, iPLA2 activities of homogenates of day 8 3T3-L1 cells after preincubation in the absence or presence of 10 µM (R)-BEL, (S)-BEL, or racemic BEL.
|
|
Pretreatment of siRNAs Targeting iPLA2 or iPLA2 Inhibits Hormone-induced Differentiation of 3T3-L1 Preadipocytes— These results, in the context of prior work on the importance of eicosanoids and lysolipids in adipocyte differentiation, suggested that iPLA2 activity may be required to promote adipocyte differentiation. To determine whether iPLA2 or iPLA2 were required for adipocyte differentiation, confluent 3T3-L1 cells were transfected with siRNA targeting iPLA2 or iPLA2. The efficiency of siRNA knockdown was judged by the iPLA2 or iPLA2 protein levels on day 4 when iPLA2s typically begin to accumulate (Fig. 4A). On day 8 of differentiation, cells were collected and the lipids were extracted for ESI/MS analysis. Treatment with siRNA directed against iPLA2 or iPLA2 largely prevented the expression of iPLA2 or iPLA2 protein. In contrast, treatment with scrambled siRNA was without effect. Remarkably, quantification of TAG using ESI/MS demonstrated that the accumulation of TAG following hormone-induced differentiation was greatly diminished after knockdown of iPLA2 or iPLA2 (Fig. 5). Next, we examined the effect of iPLA2 or iPLA2 siRNAs on several adipocyte-specific protein markers by immunoblot analysis. Western analysis demonstrated the depression of SCD-I, perilipin, GLUT 4, and PMP 70 after knockdown of iPLA2 or iPLA2 (Fig. 4B). These results indicated the requirement of iPLA2 and iPLA2 for generation of the adipocyte phenotype. To substantiate the importance of iPLA2 and iPLA2 in adipocyte differentiation utilizing an independent approach, chiral mechanism-based inhibition was employed. Treatment of 3T3-L1 cells with either (R)- or (S)-BEL substantially decreased adipocyte differentiation (Fig. 6). Interestingly, (S)-BEL is more potent than (R)-BEL in inhibiting adipogenesis (Fig. 6). This result suggests that (S)-BEL inhibits iPLA2 more potently than (R)-BEL inhibits iPLA2 and agrees with our previous in vitro assay dose-response profiles (32). Collectively, these results demonstrate the importance of both iPLA2 and iPLA2 in the adipocyte differentiation process by independent genetic and pharmacological approaches.
View larger version (42K):
[in this window]
[in a new window]
|
FIG. 4.
Effects of siRNAs directed against iPLA2 or iPLA2 on the expression of several adipocyte marker proteins. 3T3-L1 cells were cultured and transfected with 20 nM negative control siRNA (N.C.), siRNA directed against iPLA2 (), or siRNA directed against iPLA2 () prior to induction of differentiation as described under "Experimental Procedures." Total protein extracts were prepared, separated by SDS-PAGE (40 µg of protein/lane), and transferred to Immobilon-P membranes. Powdered milk (5% (w/v)) was used to block nonspecific binding sites prior to incubation with primary antibody directed against each specific protein as indicated. After incubation with HRP-conjugated secondary antibody, proteins were visualized by enhanced chemiluminescence as described under "Experimental Procedures." A, Western blot analysis of day 4 3T3-L1 cell proteins utilizing antibodies against iPLA2 or iPLA2. B, Western blot analysis of day 8 3T3-L1 cell proteins utilizing antibodies against SCD I, PMP 70, perilipin, or GLUT4.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 6.
Effects of BEL on TAG accumulation during hormone-induced differentiation of 3T3-L1 cells. 3T3-L1 cells (2 days post-confluent) were washed three times with DMEM and incubated at 37 °C for 20 min in DMEM with 0, 5, and 10 µM racemic BEL (Rac BEL), (R)-BEL, or (S)-BEL. The cells were induced to differentiate as described under "Experimental Procedures" in the presence of the indicated concentrations of BEL for 4 days. 3T3-L1 cells were grown to day 8, washed with ice-cold PBS, and scraped into 1 ml of 50 mM LiCl. The lipids were extracted by the method of Bligh and Dyer (33) in the presence of an internal standard (Tri17:0TAG, 200 nmol/mg of protein). Mass spectral analysis of TAG was performed by ESI/MS as described under "Experimental Procedures." The results represent mean ± S.E. of three independent cultures.
|
|
PPAR and C/EBP are believed to be prominent effectors of the genetic programs that induce the expression of adipocyte-specific genes leading to the development of mature adipocytes (9, 13, 35, 36). To explore the mechanism of inhibition of adipocyte differentiation imposed by knockdown of iPLA2 or iPLA2, we next examined the effect of siRNA directed against iPLA2 or iPLA2 on the expression of PPAR and C/EBP. Nuclear extracts from day 8 hormone-induced 3T3-L1 cells after pretreatment with negative control siRNA, siRNA directed against iPLA2 or siRNA directed against iPLA2 were analyzed for alterations in the expression of PPAR and C/EBP by immunoblot analysis. The expression of both PPAR and C/EBP was greatly down-regulated after transfection with iPLA2 or iPLA2 siRNAs (Fig. 7A). Thus, knockdown of iPLA2 or iPLA2 inhibited adipocyte differentiation by preventing the expression of the proadipogenic transacting factors PPAR and C/EBP.
Next, the roles of iPLA2 and iPLA2 in the hormone-induced differentiation of 3T3-L1 cells were characterized by examination of the initial induction of the early transcription factors C/EBP and C/EBP. Both C/EBP and C/EBP are essential in eliciting the expression of PPAR, which in turn leads to the induction of the expression of C/EBP (10, 37, 38). To investigate if the down-regulation of PPAR and C/EBP by silencing iPLA2 or iPLA2 was mediated by C/EBP and C/EBP, nuclear extracts from early stage hormone-induced 3T3-L1 cells pretreated with negative control siRNA, or siRNA directed against either iPLA2 or iPLA2 were prepared. Immunoblot analysis demonstrated that the transiently induced expression of C/EBP and C/EBP was not attenuated by pretreatment with siRNA directed against iPLA2 or iPLA2 (in contrast to PPAR and C/EBP) (Fig. 7B). Moreover, the transient expression of liver-enriched inhibitory protein (LIP) isoform of C/EBP, which arises from utilization of an alternative translation initiation site and is believed to be a dominant-negative regulator of C/EBP family members (39), was also not affected by siRNAs directed toward iPLA2 or iPLA2 (Fig. 7B). The lower expression levels of liver-enriched activating protein (LAP) isoform of C/EBP on day 4 after silencing iPLA2 or iPLA2 suggest that both iPLA2 and iPLA2 play roles in degradation or turnover of liver-activated protein (Fig. 7B). Because the expression levels of C/EBP decreased by over 80% on day 4 (compared with day 2), this result suggests that the temporal progression of this large decrease may be marginally delayed. Previous work has demonstrated the requirement of C/EBP for mitotic clonal expansion during adipogenesis (25, 26). The present results demonstrate that hormone-induced early stage mitotic clonal expansion was not affected by pretreatment with siRNA directed against iPLA2 or iPLA2 (Fig. 8). Collectively, these results suggest that the down-regulation of iPLA2 or iPLA2 does not prevent PPAR and C/EBP expression by affecting the expression of C/EBP and C/EBP but that these iPLA2s are essential for the activation of pathways at or proximal to the expression of PPAR and C/EBP.
Troglitazone Rescues Adipocyte Differentiation in iPLA2 or iPLA2 siRNA Pretreated 3T3-L1 Cells—To further determine whether the inhibitory effects of iPLA2 or iPLA2 siRNA on adipocyte differentiation were specifically caused by prevention of the expression and downstream effectors of PPAR and C/EBP, or alternatively if iPLA2 or iPLA2 knockdown precluded cellular differentiation by other agonists, pharmacologic activation of PPAR by troglitazone in the presence of the iPLA2 knockdowns were examined. Cultures of 3T3-L1 cells were pretreated with either siRNA against iPLA2 or siRNA against iPLA2, and incubated in differentiation media in the presence or absence of 10 µM troglitazone. On day 8 of differentiation, nuclear extracts were prepared and proteins were analyzed by immunoblotting. Troglitazone rescued the expression of PPAR and C/EBP (Fig. 9) in the presence of siRNA directed against either iPLA2 or iPLA2 and allowed completion of the differentiation process after treatment with siRNA directed against iPLA2 or iPLA2. These results support the notion that knockdown of iPLA2 or iPLA2 inhibited adipocyte differentiation by preventing the transcription programs mediated by PPAR activation and was not the result of preventing the ability of the cell to differentiate under appropriate activating conditions. Collectively, these results demonstrate that treatment of preadipocytes with siRNA directed against iPLA2 or iPLA2 can be rescued by provision of a synthetic ligand of PPAR. Because PPAR is activated by LPA derived from LPC (24), these results strongly suggest that both iPLA2 and iPLA2 can directly or indirectly provide the necessary lipid precursors for PPAR activation.
Alterations of iPLA2 and iPLA2 Expression Levels in the Zucker Obese Rat—Dysregulation of a gene in the obese state provides important clues to the functional relevance of the gene in the obese state and the mechanism contributing to obesity in that model. Up-regulation of iPLA2 and iPLA2 and the requirement of these two phospholipase proteins for adipogenesis in hormone-induced differentiation of 3T3-L1 cells suggest that they may be involved in the development of obesity. Accordingly, we investigated the modulation of iPLA2 and iPLA2 expression levels in Zucker (fa/fa) obese rats. 5-Week-old female lean and homozygous obese rats were fed ad libitum. Animals were sacrificed and inguinal fat pads (WAT) were removed for protein extraction. Protein extracts were analyzed for alterations in the expression of iPLA2 and iPLA2 by immunoblot analysis. Western blots of iPLA2 showed the dramatic up-regulation of the 65- and 40-kDa iPLA2 protein products in obese animals relative to their congenic lean controls in white adipose tissue (Fig. 10A). The identities of the 65- and 40-kDa bands were substantiated by blocking nonspecific immunoreactivity by preincubating the antibody solution with excess amounts of antigen peptide (Fig. 10A). Similarly, the expression level of the 63-kDa isoform of iPLA2 was also dramatically increased in the white adipose tissue of Zucker obese rats in comparison to that of lean control (Fig. 10B). Interestingly, the level of the 48-kDa iPLA2 proteolytic product was not altered. The identities of both 63- and 48-kDa bands were also substantiated by blocking the antibody in the presence of excess amounts of peptide antigen (Fig. 10B). Collectively, these results demonstrate the dramatic changes in iPLA2 and iPLA2 regulation in a commonly utilized genetic model of diabetes and obesity.
|
DISCUSSION
|
|---|
The present study provides multiple independent lines of evidence that iPLA2 and iPLA2 are essential regulatory components in the hormone-induced transcriptional programs that mediate the differentiation of 3T3-L1 cells into adipocytes. First, we demonstrated the up-regulation of iPLA2 and iPLA2 mRNA, protein mass, and enzymatic activity after hormone-induced differentiation of 3T3-L1 preadipocytes. Second, pharmacological inhibition of iPLA2 or iPLA2 by chiral mechanism-based inhibition resulted in the inhibition of adipocyte differentiation as assessed by the suppression of the appearance of multiple markers of mature adipocytes. Third, knockdown of iPLA2 or iPLA2 by siRNA resulted in the ablation of hormone-induced differentiation of 3T3-L1 cells as assessed by multiple independent markers of adipocyte transcriptional programs and alterations in cellular lipid content. Fourth, even in the presence of molecular biologic inhibition by siRNA knockdown, the cells could differentiate in the presence of troglitazone demonstrating that the functional integrity of processes downstream of PPAR activation was not fundamentally compromised. Collectively, these results strongly support the essential role of the iPLA2 family of enzymes in facilitating the maturation of 3T3-L1 cells into adipocytes. Because prior studies have demonstrated the importance of eicosanoid metabolites (19, 21), lysolipids (22, 24), and altered adipocyte calcium ion homeostasis (20, 40) in adipocyte differentiation, these results suggest that the iPLA2 family of enzymes serves a critical role in the provision of at least some of the lipid second messengers required for the execution of adipocyte differentiation programs.
Knockdown of iPLA2 or iPLA2 protein products inhibited the programmed expression of PPAR and C/EBP, known to be of decisive importance in the commitment to the terminal phase of adipocyte differentiation. The block appears localized distal to the production of the early transcription factors C/EBP and C/EBP and prior to the production of the late transcriptional factors PPAR and C/EBP. These results suggest that lipids produced by iPLA2 enzymes (or their downstream metabolites) modulate the transcription of PPAR and C/EBP. Moreover it seems likely that either iPLA2 or iPLA2 (or both) provides the lipids or lipid precursors that serve to activate PPAR (e.g. LPA and FFAs). Clonal expansion has generally been regarded as a prerequisite for terminal differentiation of cultured preadipocytes (26). The present study indicates iPLA2 and iPLA2 siRNAs do not interfere with the reinitiation of cell cycling of growth-arrested 3T3-L1 preadipocytes induced by differentiation inducers. Because the expression of C/EBP and C/EBP mediated mitotic clonal expansion were not affected by iPLA2 or iPLA2 siRNA pretreatment, these results localize the block in the programmed differentiation of 3T3-L1 cells into adipocytes as distal to these factors and proximal to the expression of PPAR protein. Collectively, these results identify the involvement of the signaling pathways mediated by these iPLA2s in the commitment to the terminal phase of adipocyte differentiation. Moreover, these results demonstrate that iPLA2 and iPLA2 are both required for adipocyte differentiation. iPLA2 and iPLA2 are present in different subcellular locations and are subject to distinct regulatory mechanisms potentially producing different signaling molecules at different times that are each required in appropriate temporal context for adipogenesis.
Since it was first appreciated over a decade ago, many studies have attempted to identify the lipid or lipids responsible for PPAR activation in adipocytes. Early studies demonstrated that a variety of FFAs and eicosanoids could activate the PPAR receptor (41–43). Although initial interest focused on the role of 15-deoxy-
12,14-PGJ2, the intracellular concentrations of 15-deoxy-12,14-PGJ2 are so low in comparison to their effective stimulatory concentrations for PPAR that recent studies have underscored the role of other lipids, including FFAs and LPA, as being important (24, 44). However, one cannot exclude a potential role for eicosanoids in activating PPAR, perhaps through adaptor or binding proteins that facilitate their delivery to the PPAR binding surface. At present it seems more likely that FFAs and LPA or LPA-like molecules whose functional importance have been demonstrated by a variety of molecular biologic, pharmacological, and chemical techniques are the endogenous activators of PPAR (24). Issues of concentration do not appear to be of concern with LPA as the PPAR ligand because the concentrations of LPA necessary for PPAR activation are similar to those found in biologic tissues and serum (24). Of course compartmentation and membrane surface effective concentrations are important issues that remain to be definitively addressed. Recent evidence at this point suggests the importance of the intracellular production of LPC and its subsequent hydrolysis to LPA catalyzed by a secreted adipocyte lysophospholipase D, autotaxin, or other as yet undescribed intracellular lysophospholipase D. LPA was shown to be a positive regulatory mediator of adipogenesis by interacting preferentially with the LPA1 receptor (LPA1-R) after secretion (22, 23). Moreover, expression of PPAR can be up-regulated by its activation after ligand binding. Thus, cooperative interactions between PPAR and C/EBP are likely to be present as evidenced by the fact that ectopic expression of either transcription factor alone induces the expression of the other (37, 45). This reciprocal gene activation also amplifies the effect of the PPAR ligand-mediated up-regulation of the protein expression of PPAR (feed forward activation). Finally, it should be appreciated that multiple ligands may be important and that post-translational modification of PPAR does occur. Differentially phosphorylated forms of PPAR may selectively bind to different lipids or perhaps have distinct downstream effectors depending on the nature of conformational shifts each ligand induces (46, 47). PLA2s may also regulate adipogenesis via productions of prostaglandins. PGF2 is known to be synthesized by preadipocytes and its production is dramatically decreased after induction of differentiation in 3T3-L1 cells (48). PGF2 inhibits adipocyte differentiation via activation of mitogen-activated protein kinase and subsequent phosphorylation and inhibition of PPAR (21, 48). PGE2 and PGI2, the most abundantly produced PGs by mass in adipose tissue, have differential effects on preadipocytes and adipocytes (19). PGI2 exclusively affects preadipocytes and induces adipogenesis by increasing intracellular cAMP and calcium, whereas PGE2 possesses an anti-lipolytic effect only in adipocytes (19). Collectively, these results suggest that iPLA2s exert their proadipogenic effects by providing arachidonic acid used for the production of PGE2 and PGI2 in conjunction with the direct or indirect provision of endogenous PPAR ligands (FFAs or LPA). We speculate that the production of the antiadipogenic PGF2, whose concentration decreases after induction of differentiation in 3T3-L1 cells, may be regulated by cPLA2, which dramatically decreases during the differentiation process. The results suggest that different types of PLA2 may be differentially coupled allowing production of distinct eicosanoid products in discrete subcellular pools in differentiating adipocytes.
Calcium homeostasis has been shown to play important, but complicated roles in adipocyte differentiation. Multiple reports have demonstrated an increase in intracellular calcium concentration ([Ca2+]i) during the early phase of 3T3-L1 and human preadipocyte differentiation inhibits hormone-induced adipogenesis (48, 49). Additionally, increases in [Ca2+]i during the later phase of human preadipocyte differentiation induces TAG synthesis and the expression of specific adipocyte markers (40). The results from the present study indicate that iPLA2 may provide a calcium-dependent switch in the regulation of adipocyte differentiation in response to the environmental or chemical stimuli such as adrenocorticotrophic hormone (50) and some PGs (e.g. PGF2) (48), which perturb intracellular calcium homeostasis. In this regard, important roles for iPLA2 isoforms in cellular calcium homeostasis have recently been demonstrated (51, 52). Previous work has also identified the high affinity of iPLA2 for ATP. ATP both stabilizes and activates iPLA2 isoforms and thus is a positive regulator of iPLA2 catalytic activities (53–55). Accordingly, increased ATP levels resulting from increased glycolytic flux after insulin stimulation could be a positive regulator in adipogenic signaling pathways. Thus, the notion that iPLA2 may be a sensor molecule that promotes the conversion of the excess chemical energy into lipid storage is consistent with the results of the present study. The switching from calcium-dependent cPLA2 activity to iPLA2 activity during adipocyte differentiation may have developed during evolution to sense alterations in important regulatory factors reflecting alterations in nutrient status.
Further evidence for a role of iPLA2 and iPLA2 in adipocyte development and white adipose tissue maintenance was provided by experiments utilizing genetically obese fa/fa rats. Western blot analysis demonstrated that the expression levels of iPLA2 and iPLA2 were up-regulated in homozygous Zucker obese fa/fa rats relative to their congenic lean controls in WAT. This strong up-regulation of iPLA2 and iPLA2 may contribute to the abnormal development and maintenance of WAT in these animals. It will be of interest to examine iPLA2 and iPLA2 expression levels in other obese animal models to further extend this observation.
Taken together, the present study demonstrates the disparate regulation of cPLA2 and iPLA2 classes of intracellular phospholipases during the hormone-induced differentiation of 3T3-L1 cells into adipocytes. The results identify the requirement of both iPLA2 and iPLA2 in 3T3-L1 cell differentiation into adipocytes. It is now clear that increases in adipogenesis contribute to the development of obesity by increasing the number of mature adipocytes in multiple mammalian models. Thus, the present results identify a potential in vivo role for iPLA2s in the regulation of obesity and the related pathophysiologic sequelae of the metabolic syndrome.
|
FOOTNOTES
|
|---|
* This work was supported by National Institutes of Health Grants 5P01HL57278-07 and RO1DK59577. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
** To whom correspondence should be addressed: 660 South Euclid Ave., Campus Box 8020, St. Louis, MO 63110. Tel.: 314-362-2690; Fax: 314-362-1402; E-mail: dwamser{at}pcg.wustl.edu.
1 The abbreviations used are: WAT, white adipose tissue; PLA2, phospholipases A2; iPLA2, calcium-independent phospholipase A2; cPLA2, cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; TAG, LPA, lysophosphatidic acid; LPC, lysophosphatidylcho-triacylglycerol; line; FFA, free fatty acid; PG, prostaglandin; siRNA, small interfering RNA; ESI/MS, electrospray ionization mass spectrometry; PPAR, peroxisome proliferator-activated receptor ; C/EBP, CCAAT enhancer binding protein; BEL, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
|
REFERENCES
|
|---|
- Friedman, J. M. (2000) Nature 404, 632–634[Medline]
[Order article via Infotrieve]
- Spiegelman, B. M., and Flier, J. S. (2001) Cell 104, 531–543[CrossRef][Medline]
[Order article via Infotrieve]
- Gregoire, F. M., Smas, C. M., and Sul, H. S. (1998) Physiol. Rev. 78, 783–809[Abstract/Free Full Text]
- Friedman, J. M., and Halaas, J. L. (1998) Nature 395, 763–770[CrossRef][Medline]
[Order article via Infotrieve]
- Steppan, C. M., Bailey, S. T., Bhat, S., Brown, E. J., Banerjee, R. R., Wright, C. M., Patel, H. R., Ahima, R. S., and Lazar, M. A. (2001) Nature 409, 307–312[CrossRef][Medline]
[Order article via Infotrieve]
- Moustaid, N., and Sul, H. S. (1991) J. Biol. Chem. 266, 18550–18554
TOP
ABSTRACT
INTRODUCTION
