Phosphorylation of GATA2 by Akt Increases Adipose Tissue Differentiation and Reduces Adipose Tissue–Related Inflammation
http://www.100md.com
循环学杂志 2005年第4期
the Departments of Internal Medicine (R.M., V.M., M.C., M.L.H., D.L., P.S., R.L., M.F.) and Biopathology and Diagnostic Imaging (A.M.), University of Rome Tor Vergata
Center for Atherosclerosis (D.L., P.S., R.L., M.F.), Policlinico Tor Vergata–PTV University Hospital, Rome, Italy.
Abstract
Background— Obesity-related inflammation is emerging as a major cause of insulin resistance and cardiovascular diseases. GATA2 transcription factor is an inhibitor of adipogenesis and an activator of vascular cells. We hypothesized that GATA2 activity is controlled by insulin during adipogenesis, linking metabolic homeostasis and inflammation.
Methods and Results— We show that insulin induces GATA2 phosphorylation on serine 401 in a PI-3K/Akt–dependent manner. Insulin-dependent phosphorylation of serine 401 impairs GATA2 translocation to the nucleus and its DNA binding activity. A GATA2 mutant not phosphorylable by Akt (GATA2S401A) acts similarly to wild-type GATA2. In contrast, a GATA2 mutant that mimics Akt phosphorylation (GATA2S401D) is restrained in the cytoplasm. Cultured preadipocytes bearing GATA2S401A do not convert to adipocytes and express high levels of inflammatory cytokines like monocyte chemotactic protein-1 (MCP-1). On the contrary, GATA2S401D preadipocytes differentiate to adipocytes. When GATA2S401A preadipocytes are injected in mice fed a high-fat diet, they do not differentiate adequately into adipocytes, maintaining the expression of inflammatory markers like MCP-1. In contrast, injection of GATA2S401D preadipocytes in mice fed a high-fat diet results in development of adipocytes and no expression of inflammatory markers.
Conclusions— GATA2 could be a new target in the prevention and treatment of obesity-related inflammation and its complications.
Key Words: diabetes mellitus ; inflammation ; insulin ; obesity ; signal transduction
Introduction
Obesity, a major cause of cardiovascular diseases and type 2 diabetes, is caused by an expansion of white adipose tissue upon alteration of energy balance.1 Recently, it has been observed that adipocyte precursors and immunocompetent cells like macrophages share similar functions.2–4 This link has been strengthened by the observation that both animal models of obesity and human obesity are characterized by accumulation among adipocytes of monocyte/macrophages, which in turn would be the main source of proinflammatory cytokines and a key cause of adipose-specific and systemic insulin resistance.5,6 The failure of adipocytes to adapt to the demands of energy storage is associated with the reduced production of antiatherogenic hormones like adiponectin and increased secretion of proinflammatory cytokines such as tumor necrosis factor- (TNF-).7,8 Interestingly, an inverse relationship between adipose cell and macrophage differentiation has been described. Thus, it appears that when differentiation into adipocytes is enhanced, an inhibition of macrophage activation occurs.3 However, mechanisms explaining macrophage accumulation among adipocytes have not been clarified, and factors modulating metabolic versus inflammatory behavior of preadipocytes are unknown.9 Preadipocytes, located in the stromal vascular fraction of adipose tissue, differentiate under appropriate hormonal and nutritional stimuli.10 Upon differentiation, they acquire a different phenotype and become able to store energy through synthesis of lipids.1,11 Insulin is a major stimulator of adipogenesis through the activation of a pathway involving insulin receptor (IR), IR substrate-1 (IRS-1), and the downstream effectors phosphatidylinositol 3-kinase (PI-3K) and Akt.11–14 It has been suggested that Akt controls adipogenesis and metabolic homeostasis in part by modulation of transcription factor activity, as is the case for FOXO1.15 Thus, when insulin resistance occurs, it causes impaired activity of the PI-3K/Akt pathway, resulting in inadequate adipogenesis and adipose tissue metabolism. Interestingly, adipogenesis is blocked by GATA2, a transcription factor specifically expressed in the stromal vascular fraction of the adipose tissue where preadipocytes reside. In fact, GATA2 blocks the transition from preadipocyte to adipocyte in part through suppression of PPAR-2 expression.16 GATA2 is also known to direct hematopoiesis and vascular cell activation.17–19 Therefore, we hypothesized that in preadipocytes inhibition of GATA2 could allow adipogenesis while reducing their inflammatory properties. An Akt consensus motif was reported to be common to GATA1, GATA2, and GATA3 and conserved among human, rat, and mouse.20 We reasoned that insulin could modulate GATA2 activity similarly to FOXO factors.15
Here, we present evidence that GATA2 is phosphorylated and blocked by the PI3K/Akt signal transduction pathway. GATA2 phosphorylation results in preadipocyte conversion to adipocytes and attenuation of the inflammatory behavior of preadipocytes. Our findings reveal a new scenario for the relationship between obesity and inflammation, suggesting a potential pathway to hamper the inflammatory state characterizing obesity and atherosclerosis.
Methods
Expression Plasmids
Human GATA2 (hGATA2) cDNA was kindly provided by Gianluigi Condorelli (University of California, San Diego). Expression vector for hGATA2 was generated by cloning the coding region in pCMV-HA vector containing an N-terminal hemaglutinin (HA) epitope tag (Clontech). Mutants (GATA2S401A, in which Ser401 was replaced by an alanine, and GATA2S401D, in which Ser401 was replaced by an aspartate) were generated by PCR mutagenesis using the Quikchange protocol (Stratagene). The mutations were confirmed by DNA sequencing. Myc-tagged constitutively active (caAKT) or inactive (dnAKT) form of Akt in pUSE were from Upstate Biotech. Wild-type hIR cDNA was subcloned in pCO11 expression vector. Retroviral plasmids were constructed by cloning the coding regions in pLXSN retroviral vector (Clontech).
Immunoblotting and Immunoprecipitation
Human embryo kidney cells (HEK293) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 100 U/mL penicillin/streptomycin (Invitrogen). Transient transfections were performed with calcium phosphate using a standard protocol.21 Cells were serum starved 18 hours before treatment with/without insulin (Sigma) and wortmannin (Sigma) or rapamicin (Sigma). Immunoblot experiments were performed as previously described,22 with anti–phospho-(Ser/Thr)Akt substrate Ab (cell signaling) or anti–HA-Tag polyclonal Ab (Clontech).
In Vitro Akt Kinase Assay
HEK293 cells were transiently transfected with either a constitutively active (caAKT) or an inactive form (dnAKT) of Akt (Upstate Biotech.) or HA-GATA2 constructs. The immunoprecipitates were combined and incubated in Akt kinase assay buffer, and the reaction was terminated as described previously.22 Samples were subjected to 8% SDS-PAGE, and phosphorylated GATA2 was detected with a PhosphoImager. In addition, the blot was reprobed with anti–phospho-(Ser/Thr)Akt substrate Ab.
In Vivo Phosphorylation
HEK293 cells were transiently transfected, serum starved 18 hours, and then labeled for 4 hours with [P32]-orthophosphate (75 μCi/mL in KRB buffer, pH 7,4, containing 1% BSA). After labeling, cells were treated without or with insulin, washed 4 times with PBS, and lysed. Lysates were immunoprecipitated with anti-HA antibody; samples were separated by 8% SDS-PAGE and transferred to nylon membrane. Phosphorylated GATA2 was detected with a PhosphoImager, and the filter was reprobed with anti–HA-Tag polyclonal Ab to normalize the transfection levels.
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from HEK293-transfected cells, and GATA2 transcriptional activity was determined by use of an electrophoretic mobility shift assay (EMSA) as described.23 In addition, nuclear extracts were separated by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti–HA-Tag polyclonal Ab (Clontech).
Immunofluorescence
SAOS-1 human osteosarcoma cells were grown as described for HEK293 and plated on coverslips in 6-well plates. Cells were transfected with LipofectAMINE-2000 (Invitrogen). After being rinsed 2 times with PBS, cells were fixed in 4% paraformaldehyde, pH 7.4, for 20 minutes and permeabilized with 0.05% Triton X100 for 2 minutes. Coverslips were incubated for 1 hour with anti–HA-Tag polyclonal Ab and then for 1 hour with a fluorescein-conjugated anti-rabbit IgG (Jackson ImmunoResearch), and nuclei were stained with 4-6-diamidino-2-phenylindole-2HCl (DAPI, Sigma). Coverslips were examined by laser confocal microscopy (Nikon). For quantification, 250 cells per coverslip were counted.
Infection and Oil Red O Staining
Packaging of the retroviral particles was achieved by transfecting the LXSN expression plasmids into EcoPack-293 packaging cell line (Clontech) with the Fugene 6 reagent (Roche) according to the protocol. Forty-eight hours after transfection, supernatants from packaging cells were collected and filtered through sterile 0.45-μm syringe filters. Twenty-four hours before infection, 3T3F442A mouse preadipocyte cells (Ecacc) were seeded at a density of 2x105 per 60-mm plates. For infection, recipient cells were incubated with viral supernatant containing a final concentration of 4 μg/mL Polybrene (Sigma). After 5 hours, cells were fed with fresh Dulbecco’s modified Eagle’s medium supplemented with 10% newborn calf serum and 100 U/mL penicillin/streptomycin (Gibco) and allowed to grow to 80% confluence. Once confluent cells were differentiated, they were stained with oil red O solution.15 Cells were kept in the presence of the appropriate antibiotic throughout the experiments to maintain stable expression levels of GATA2, GATA2S401A, and GATA2S401D.
RNA Isolation, Reverse-Transcriptase Polymerase Chain Reaction, and Northern Blotting
Total RNA was isolated with TRIzol reagent (Life Technologies) as described by the manufacturer. For reverse-transcriptase polymerase chain reaction (RT-PCR), 1 μg total RNA was reverse-transcribed into cDNA with 1 U/mL of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen) at 42°C for 45 minutes. Primer sequences are available on request. Northern hybridization was performed according to standard techniques.15
Cytokines Antibody Array
Protein extracts (250 μg) from GATA2 mutant–infected 3T3-F442A preadipocytes were incubated with the mouse cytokine antibody array membranes according to the protocol (Panomics, Inc). The active cytokines were visualized by chemiluminescence.
Phagocytosis Studies
Phagocytosis assay was performed by incubating cells with fluorescently labeled zymosan A (S cerevisiae) bioparticles that were fluorescein conjugated (Molecular Probes).3 Nuclei were stained with DAPI 2 μg/mL in PBS buffer. Phagocytic activity was identified with a confocal microscopy (Nikon) and reported as percentage of phagocytizing cells.
Flow Cytometry Analysis
For direct immunofluorescence by flow cytometry, nonconfluent (day –1) 3T3-F442A was analyzed for the expression of F4/80 with F4/80-PE antibody (Caltag Laboratories Inc) as described.5
Subcutaneous Implantation of Preadipocytes, Excision of Fat Pads, and Histology
Infected 3T3-F442A preadipocytes were injected subcutaneously (3x107 cells per site) into the back of athymic mice (Harlan). Seven weeks later, the resulting tissue was dissected and analyzed as described.24 Total RNA was isolated by use of the TRIzol reagent from a portion of dissected tissue and analyzed by RT-PCR as described above.
Statistical Analysis
Data are expressed as mean±SD. Statistical analyses were performed with ANOVA or Student t test as indicated.
Results
Akt Phosphorylates GATA2 In Vitro at Ser401
To analyze the interaction between Akt and GATA2, we first determined whether GATA2 could be phosphorylated by Akt in vitro. We generated HA epitope-tagged vectors containing either a GATA2 wild-type or a nonphosphorylable form in which the putative Akt phosphorylation site at Ser401 was replaced with a nonphosphorylable alanine residue (GATA2S401A). HEK293 cells were transfected with HA-GATA2 wild-type or HA-GATA2S401A, and the 2 variants of GATA2 were immunoprecipitated with an anti–HA-Tag antibody. The immunoprecipitates were combined with a constitutively active form of Akt (caAkt) in the presence of [-32P]ATP to perform an in vitro kinase assay and separated by SDS-PAGE. Autoradiograph showed that caAkt was able to phosphorylate wild-type GATA2 but not GATA2S401A (Figure 1A, top). In contrast, an inactive form of Akt (dnAkt) was not able to phosphorylate HA-GATA2 wild-type or HA-GATA2S401A, indicating that Ser401 of GATA2 is likely to be an Akt target (Figure 1A, top). To confirm that Ser401 is an Akt phosphorylation site in vitro, we analyzed the same membrane by Western blot with anti–Akt-phospho substrate Ab, which specifically recognizes amino acid sequences containing a serine or threonine residue phosphorylated by Akt. We confirmed that only wild-type GATA2 is phosphorylated by caAkt (Figure 1A, middle). These results raise the possibility that GATA2 may be a novel substrate for Akt.
Phosphorylation of GATA2 at Ser401 in Response to Insulin
Next, we tested the hypothesis that insulin stimulates GATA2 Ser401 phosphorylation in living cells. For this experiment, we used HEK293 cells, which are an optimal model for transfection assays. Cells transfected with HA-GATA2, HA-GATA2S401A, or HA empty vector were labeled with [32P]orthophosphate and stimulated with insulin for 5, 15, or 30 minutes. Thereafter, GATA2 was immunoprecipitated with an anti–HA-Tag Ab and separated by SDS-PAGE. We found that insulin induced a significant phosphorylation of GATA2 after 30 minutes of treatment, whereas in GATA2S401A-transfected cells, we did not observe any increase in 32P content (Figure 1B). These data indicate that GATA2 is phosphorylated at Ser401 in an insulin-dependent manner. We next investigated whether GATA2 phosphorylation is regulated by the insulin/IR/Akt signaling pathway. To increase activation of the insulin/IR/Akt signaling pathway, cells were cotransfected with either HA-GATA2 or HA-GATA2S401A and IR vectors and stimulated with insulin. GATA2 and GATA2S401A were immunoprecipitated and separated by SDS-PAGE, and phosphorylation levels were detected by Western blotting with an anti–Akt-phospho substrate antibody. Phosphorylation was already induced after 10 minutes in GATA2-transfected cells, but not in cells transfected with GATA2S401A (Figure 1C), indicating that insulin-dependent phosphorylation of Ser401 is activated by Akt in an insulin receptor dependent manner.
Phosphorylation of GATA2 at Ser401 in Response to Insulin Is Inhibited by Wortmannin
To determine whether insulin-dependent phosphorylation of GATA2 is directly mediated by the IR/PI3K/Akt signaling pathway, we performed phosphorylation experiments in the presence of wortmannin, an inhibitor of PI 3-kinase activity. For these experiments, HEK293 cells were cotransfected with either HA-GATA2 or HA-GATA2S401A and IR vectors. Pretreatment of cells with wortmannin abolished the ability of insulin to stimulate phosphorylation of GATA2 (Figure 1D). Because it was previously shown that GATA factors might be phosphorylated by mTOR and MAPK, we investigated whether these pathways could affect Ser401 phosphorylation.25,26 Neither the mTOR inhibitor rapamycin (Figure 1D) nor the MAPK inhibitor PD098059 (data not shown) affect insulin-dependent GATA2 phosphorylation at Ser401. These results further support a role for Akt in the phosphorylation of GATA2 at Ser401.
Role of Ser401 in Regulating Function of GATA2
Next, we tested the hypothesis that insulin impairs the transactivation potential of GATA2 through phosphorylation at Ser401 by Akt. We analyzed the effect of insulin on GATA2 interaction with specific DNA sequences by an EMSA assay. We found that insulin reduced the GATA2-DNA complex in HEK293 cells transfected with HA-GATA2 (Figure 2A). Immunoblotting with the anti–HA-Tag antibody performed on the same extracts showed that insulin reduced the nuclear amount of GATA2 protein (Figure 2B).
To determine whether phosphorylation at Ser401 is involved in the regulation of the binding activity and nuclear protein amount, we analyzed the effects of insulin in cells transfected either with GATA2S401A or with a mutant in which Ser401 was replaced with aspartate (GATA2S401D), which mimics a constitutive phosphorylation state. We found that GATA2S401A was bound to the DNA sequences in either presence or absence of insulin. Instead, the complex formation was inhibited in cells transfected with the HA-GATA2S401D vector (Figure 2C). A Western blot assay performed with nuclear extracts from the same cells showed that the nuclear protein expression of GATA2S401D was dramatically reduced with respect to GATA2S401A (Figure 2D). These results suggest that phosphorylation of GATA2 at Ser401 modulates its intracellular localization.
Subcellular Localization of GATA2 Mutants
Then, we asked whether phosphorylation of Ser401 affects the subcellular localization of GATA2. For this experiment, we used SAOS-1 cells because they are easily transfectable and, having a good nucleus to cytoplasm proportion, they allow optimal visualization of cytoplasm/nucleus trafficking. We transiently transfected these cells with either HA-GATA2S401A or HA-GATA2S401D and performed confocal immunofluorescence analysis with an anti–HA-Tag antibody. We observed that GATA2S401A was localized to both nucleus and cytoplasm, whereas GATA2S401D was prevalently cytoplasmatic (Figure 2E). Taken together, these results suggest that the insulin-induced phosphorylation at Ser401 modulates GATA2 DNA binding activity because it reduces the nuclear amount of the protein.
Role of GATA2 Ser401 in Adipocyte Differentiation In Vitro
GATA2 is involved in the regulation of the preadipocyte-adipocyte differentiation. Thus, to investigate the physiological role of Ser401 phosphorylation in preadipocyte-adipocyte differentiation, we took advantage of 3T3-F442A preadipocyte cells, a widely used culture model for adipogenesis. Because the differentiation process is extended for 12 to 14 days in 3T3-F442A, we elected to use retroviral constructs to introduce and maintain GATA2 mutant expression in these cells. Retrovirus-infected preadipocytes are known to retain a good level of expression through differentiation.10 Therefore, we generated retroviral constructs encoding GATA2 (LX-GATA2), GATA2S401A (LX-GATA2S401A), and GATA2S401D (LX-GATA2S401D) and used them to produce various 3T3-F442A clones. We also infected 3T3-F442A with the empty vector (LXSN, mock) for control. We confirmed by RT-PCR that expression of GATA2 wild-type and mutant constructs was maintained during differentiation for up to 16 days (Figure 3A). Subsequently, we analyzed the ability of the infected clones to differentiate. Adipogenesis was normal in 3T3-F442A mock-infected cells. In contrast, both GATA2 and GATA2S401A exerted an inhibitory effect on intracellular lipid accumulation, whereas 3T3-F442A infected with LX-GATA2S401D exhibited normal adipogenesis, as assessed by oil red O staining at day 16 (Figure 3B). Northern blot analysis showed a marked decreased in PPAR- expression in 3T3-F442A infected with LX-GATA2 and LX-GATA2S401A compared with LX-GATA2S401D– and mock-infected 3T3-F442A cells (Figure 3C). Thus, phosphorylation of GATA2 on Ser401 appears to be crucial for the adipogenesis program in vivo.
Role of GATA2 Ser401 in Phagocytic Activity
Preadipocytes exhibit phagocytic activity similarly to macrophages.3 An inverse relationship between adipose cell and macrophage differentiation has been described.3 To determine whether the phosphorylation of GATA2 on Ser401 could affect common functions between preadipocytes and macrophages, we measured phagocytic activity in growing preadipocytes infected with GATA2 mutants. LX-GATA2– and LX-GATA2S401A–infected cells showed a significantly increased phagocytosis with respect to the LX-GATA2S401D and control cells (Figure 4A), indicating that the phosphorylation of GATA2 on Ser401 is involved in the link between adipose tissue and immunity processes.
Many mediators of inflammation are secreted by adipocytes. To investigate whether the 2 mutants, GATA2S401A and GATA2S401D, display a different profile of expression of inflammatory proteins, we used a cytokine protein array. Cell lysates from LX-GATA2S401A– or LX-GATA2S401D–infected 3T3F442A preadipocytes were incubated with arrays containing antibodies directed against various inflammatory proteins. We found that cells infected with LX-GATA2S401A express significantly higher levels of macrophagelike cytokines such as monocyte chemotactic protein-1 (MCP-1), thrombopoietin (TPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 4 (IL-4) compared with cells infected with LX-GATA2S401D (Figure 4B). We confirmed the upregulation of MCP-1 in GATA2S401A with respect to GATA2S401D by RT-PCR (Figure 4C). In contrast, analysis by both RT-PCR (Figure 4C) and flow cytometry (data not shown) of F4/80, a marker for mature macrophages, was negative in 3T3-F442A cells infected with LXSN, LX-GATA2, LX-GATA2S401A, and LX-GATA2S401D (Figure 4C) and in differentiated 3T3-F442A adipocytes (data not shown).
Role of GATA2 Ser401 in Adipocyte Differentiation In Vivo
Next, we performed experiments to assess the role of GATA2 phosphorylation at Ser401 in animal models. Implanted 3T3-F442A preadipocytes injected into nude mice fed a high-fat diet gave rise to fat pads indistinguishable from normal adipose tissue.24 To examine whether the effects of Ser401 phosphorylation are maintained in vivo, we subcutaneously injected into athymic mice 3T3-F442A preadipocytes infected with the different LX-GATA2 constructs or LXSN. Mice were fed a high-fat diet to trigger adipocyte differentiation. Seven weeks later, we dissected the resulting tissue. We found that preadipocytes infected with LXSN and LX-GATA2S401D were able to differentiate into adipocytes, whereas cells bearing LX-GATA2 and LX-GATA2S401A formed pads composed mostly of undifferentiated fibroblastlike cells and rare adipocytes (Figure 5A). To confirm that the tissues were derived from the injected preadipocytes, we assessed hGATA2 expression by RT-PCR (Figure 5B). All fat pads were positive for hGATA2 expression. The area covered by adipocytes in the LX-GATA2S401D fat pad was 5-fold higher than LX-GATA2S401A (P<0.01; data not shown). Moreover, LX-GATA2S401D–infected cells formed both unilocular and multilocular adipocytes, which were not observed in LX-GATA2S401A fat pads.
Because we found increased expression of MCP-1 in cultured preadipocytes infected with retrovirus encoding GATA2 and GATA2S401A, we evaluated mRNA expression of mouse MCP-1 and F4/80 in fat pads (Figure 5B). Both MCP-1 and F4/80 mRNA were upregulated in fat pads derived from implants of 3T3-F442A cells infected with GATA2 and GATA2S401A (Figure 5B).
Discussion
Obesity-related inflammation is emerging as a cause of systemic insulin resistance and atherosclerosis. Inflammation plays a major role in the development of cardiovascular events. In fact, advanced or unstable atherosclerotic plaques are known to be in an even higher state of inflammation than stable plaques. Thus, obesity has been hypothesized to be a proinflammatory state, increasing the vulnerability of atherosclerotic plaques. However, mechanisms explaining how adiposity triggers inflammation are still elusive.27 It is recognized that adipose and hematopoietic tissues share some characteristics. In fact, some in vitro studies showed that preadipocytes could behave like or even transform into macrophages.3,4 More recently, a specific accumulation of macrophages in adipose tissue has been demonstrated. Inflammatory markers would derive only from these macrophages, which would be responsible for the effects on insulin sensitivity and vascular homeostasis.5,6 However, it remains unclear which factors balance the preadipocyte fate toward an adipocyte or macrophage phenotype, as well as which signals drive monocyte/macrophage accumulation into adipose tissue.
Adipose tissue function is controlled by hormonal cues that, through signals delivered to the nucleus, modulate the transcription of enzymes and molecules that regulate lipid metabolism.11
Our results show that GATA2 is a novel Akt-regulated transcription factor involved in adipogenesis. In addition, our findings suggest that the Akt/GATA2 axis could act as a balance between the adipogenic and the macrophagelike phenotype of preadipocytes. GATA2 when phosphorylated by Akt is excluded from the nucleus, and preadipocytes differentiate, losing their ability to exert inflammatory functions. In contrast, when GATA2 is inadequately phosphorylated, it blocks adipogenesis by inhibiting PPAR- expression, thus maintaining preadipocytes in a macrophagelike state. In fact, preadipocytes expressing wild-type GATA2 and GATA2S401A show increased phagocytic behavior compared with preadipocytes bearing GATA2S401D. This is consistent with previous observations suggesting that GATA2 plays a major role in phagocytosis. In fact, macrophages infected with P carinii lose GATA2 expression and consequently phagocytic activity. Furthermore, once GATA2 is overexpressed in infected macrophages, they reacquire phagocytic activity.28 Therefore, our results corroborate the observation that preadipocytes behave like macrophages.3 However, we observed that preadipocytes expressing GATA2 do not bear a typical macrophage marker like F4/80, indicating that they are not fully converted into macrophages. Our data support the hypothesis that GATA2 activity in these preadipocytes takes part in the complex regulation of monocyte/macrophage trafficking, generating specific signals necessary to drive monocyte accumulation among adipocytes.9 In fact, we show that in preadipocytes an unchecked GATA2 (GATA2S401A) leads to increased MCP-1 and GM-CSF expression, while the opposite is seen when GATA2 activity is inhibited (GATA2S401D). Noteworthy, both MCP-1 and GM-CSF play a role in monocyte diapedesis, although at different extents.29,30 Our results are compatible with recent observations suggesting that the inflammatory burden of adiposity is due to macrophage cell accumulation in the adipose tissue.6,9 Moreover, because GATA2 is involved in vascular cell adhesion molecule-1 expression, an adhesion molecule determinant for monocyte adhesion to other cells,31 it is plausible that GATA2 action in endothelial cells could facilitate monocyte diapedesis in the vessel wall, accounting also for a possible proatherosclerotic effect of this transcription factor.
To determine the role of GATA2 on adipogenesis in vivo, we used the fat pad formation model. In mice injected with mock-infected or GATA2S401D-infected cells, adipocytes develop normally. In contrast, in mice injected with preadipocytes bearing an unchecked GATA2, fat pad formation is blocked, and we observed increased mRNA expression of both MCP-1, confirming our in vitro observation, and F4/80. Because F4/80 was not expressed by preadipocytes in vitro, it is conceivable that it derives from monocytes that migrated near injected cells in response to the increased MCP-1 expression.
We can speculate that insulin triggers adipogenesis to maintain metabolic homeostasis by restraining in preadipocytes both repressors of metabolic program like FOXO-1, as well as factors regulating cell cycle and inflammatory properties like GATA2. Therefore, in insulin resistance, unchecked FOXO1 might inhibit energy storage, causing metabolic derangement.15 Simultaneously, unchecked GATA2 might facilitate monocyte homing and macrophage differentiation via an increased expression of factors such as MCP-1, GM-CSF, and IL-4 from preadipocytes.29–32 Interestingly, a synthetic inhibitor for GATA2 has recently been developed and shown to act as an antiinflammatory agent.33
Our data suggest that insulin regulating GATA2 can direct preadipocytes toward an adipogenic program, whereas insulin resistance, resulting from either genetic or metabolic factors, would facilitate a GATA2-sustained inflammatory program. Therefore, the inhibition of GATA2 could be investigated as a new strategy to lower obesity-related inflammation to prevent the development of late complications of obesity such as diabetes and plaque rupture.
Acknowledgments
This study was supported by grants from the Italian Ministry of University (PRIN 2003067733-001 to Dr Lauro; PRIN 2003067733-006 to Dr Federici), Italian Ministry of Health (RFS 2001, 2002, and 2003 to Dr Federici), and a grant to University of Tor Vergata Center of Excellence on Genomic Risk Assessment (MIUR CE00277884 to Dr Lauro). Drs Menghini and Hribal are supported by a grant from the University of Tor Vergata.
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Center for Atherosclerosis (D.L., P.S., R.L., M.F.), Policlinico Tor Vergata–PTV University Hospital, Rome, Italy.
Abstract
Background— Obesity-related inflammation is emerging as a major cause of insulin resistance and cardiovascular diseases. GATA2 transcription factor is an inhibitor of adipogenesis and an activator of vascular cells. We hypothesized that GATA2 activity is controlled by insulin during adipogenesis, linking metabolic homeostasis and inflammation.
Methods and Results— We show that insulin induces GATA2 phosphorylation on serine 401 in a PI-3K/Akt–dependent manner. Insulin-dependent phosphorylation of serine 401 impairs GATA2 translocation to the nucleus and its DNA binding activity. A GATA2 mutant not phosphorylable by Akt (GATA2S401A) acts similarly to wild-type GATA2. In contrast, a GATA2 mutant that mimics Akt phosphorylation (GATA2S401D) is restrained in the cytoplasm. Cultured preadipocytes bearing GATA2S401A do not convert to adipocytes and express high levels of inflammatory cytokines like monocyte chemotactic protein-1 (MCP-1). On the contrary, GATA2S401D preadipocytes differentiate to adipocytes. When GATA2S401A preadipocytes are injected in mice fed a high-fat diet, they do not differentiate adequately into adipocytes, maintaining the expression of inflammatory markers like MCP-1. In contrast, injection of GATA2S401D preadipocytes in mice fed a high-fat diet results in development of adipocytes and no expression of inflammatory markers.
Conclusions— GATA2 could be a new target in the prevention and treatment of obesity-related inflammation and its complications.
Key Words: diabetes mellitus ; inflammation ; insulin ; obesity ; signal transduction
Introduction
Obesity, a major cause of cardiovascular diseases and type 2 diabetes, is caused by an expansion of white adipose tissue upon alteration of energy balance.1 Recently, it has been observed that adipocyte precursors and immunocompetent cells like macrophages share similar functions.2–4 This link has been strengthened by the observation that both animal models of obesity and human obesity are characterized by accumulation among adipocytes of monocyte/macrophages, which in turn would be the main source of proinflammatory cytokines and a key cause of adipose-specific and systemic insulin resistance.5,6 The failure of adipocytes to adapt to the demands of energy storage is associated with the reduced production of antiatherogenic hormones like adiponectin and increased secretion of proinflammatory cytokines such as tumor necrosis factor- (TNF-).7,8 Interestingly, an inverse relationship between adipose cell and macrophage differentiation has been described. Thus, it appears that when differentiation into adipocytes is enhanced, an inhibition of macrophage activation occurs.3 However, mechanisms explaining macrophage accumulation among adipocytes have not been clarified, and factors modulating metabolic versus inflammatory behavior of preadipocytes are unknown.9 Preadipocytes, located in the stromal vascular fraction of adipose tissue, differentiate under appropriate hormonal and nutritional stimuli.10 Upon differentiation, they acquire a different phenotype and become able to store energy through synthesis of lipids.1,11 Insulin is a major stimulator of adipogenesis through the activation of a pathway involving insulin receptor (IR), IR substrate-1 (IRS-1), and the downstream effectors phosphatidylinositol 3-kinase (PI-3K) and Akt.11–14 It has been suggested that Akt controls adipogenesis and metabolic homeostasis in part by modulation of transcription factor activity, as is the case for FOXO1.15 Thus, when insulin resistance occurs, it causes impaired activity of the PI-3K/Akt pathway, resulting in inadequate adipogenesis and adipose tissue metabolism. Interestingly, adipogenesis is blocked by GATA2, a transcription factor specifically expressed in the stromal vascular fraction of the adipose tissue where preadipocytes reside. In fact, GATA2 blocks the transition from preadipocyte to adipocyte in part through suppression of PPAR-2 expression.16 GATA2 is also known to direct hematopoiesis and vascular cell activation.17–19 Therefore, we hypothesized that in preadipocytes inhibition of GATA2 could allow adipogenesis while reducing their inflammatory properties. An Akt consensus motif was reported to be common to GATA1, GATA2, and GATA3 and conserved among human, rat, and mouse.20 We reasoned that insulin could modulate GATA2 activity similarly to FOXO factors.15
Here, we present evidence that GATA2 is phosphorylated and blocked by the PI3K/Akt signal transduction pathway. GATA2 phosphorylation results in preadipocyte conversion to adipocytes and attenuation of the inflammatory behavior of preadipocytes. Our findings reveal a new scenario for the relationship between obesity and inflammation, suggesting a potential pathway to hamper the inflammatory state characterizing obesity and atherosclerosis.
Methods
Expression Plasmids
Human GATA2 (hGATA2) cDNA was kindly provided by Gianluigi Condorelli (University of California, San Diego). Expression vector for hGATA2 was generated by cloning the coding region in pCMV-HA vector containing an N-terminal hemaglutinin (HA) epitope tag (Clontech). Mutants (GATA2S401A, in which Ser401 was replaced by an alanine, and GATA2S401D, in which Ser401 was replaced by an aspartate) were generated by PCR mutagenesis using the Quikchange protocol (Stratagene). The mutations were confirmed by DNA sequencing. Myc-tagged constitutively active (caAKT) or inactive (dnAKT) form of Akt in pUSE were from Upstate Biotech. Wild-type hIR cDNA was subcloned in pCO11 expression vector. Retroviral plasmids were constructed by cloning the coding regions in pLXSN retroviral vector (Clontech).
Immunoblotting and Immunoprecipitation
Human embryo kidney cells (HEK293) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 100 U/mL penicillin/streptomycin (Invitrogen). Transient transfections were performed with calcium phosphate using a standard protocol.21 Cells were serum starved 18 hours before treatment with/without insulin (Sigma) and wortmannin (Sigma) or rapamicin (Sigma). Immunoblot experiments were performed as previously described,22 with anti–phospho-(Ser/Thr)Akt substrate Ab (cell signaling) or anti–HA-Tag polyclonal Ab (Clontech).
In Vitro Akt Kinase Assay
HEK293 cells were transiently transfected with either a constitutively active (caAKT) or an inactive form (dnAKT) of Akt (Upstate Biotech.) or HA-GATA2 constructs. The immunoprecipitates were combined and incubated in Akt kinase assay buffer, and the reaction was terminated as described previously.22 Samples were subjected to 8% SDS-PAGE, and phosphorylated GATA2 was detected with a PhosphoImager. In addition, the blot was reprobed with anti–phospho-(Ser/Thr)Akt substrate Ab.
In Vivo Phosphorylation
HEK293 cells were transiently transfected, serum starved 18 hours, and then labeled for 4 hours with [P32]-orthophosphate (75 μCi/mL in KRB buffer, pH 7,4, containing 1% BSA). After labeling, cells were treated without or with insulin, washed 4 times with PBS, and lysed. Lysates were immunoprecipitated with anti-HA antibody; samples were separated by 8% SDS-PAGE and transferred to nylon membrane. Phosphorylated GATA2 was detected with a PhosphoImager, and the filter was reprobed with anti–HA-Tag polyclonal Ab to normalize the transfection levels.
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from HEK293-transfected cells, and GATA2 transcriptional activity was determined by use of an electrophoretic mobility shift assay (EMSA) as described.23 In addition, nuclear extracts were separated by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti–HA-Tag polyclonal Ab (Clontech).
Immunofluorescence
SAOS-1 human osteosarcoma cells were grown as described for HEK293 and plated on coverslips in 6-well plates. Cells were transfected with LipofectAMINE-2000 (Invitrogen). After being rinsed 2 times with PBS, cells were fixed in 4% paraformaldehyde, pH 7.4, for 20 minutes and permeabilized with 0.05% Triton X100 for 2 minutes. Coverslips were incubated for 1 hour with anti–HA-Tag polyclonal Ab and then for 1 hour with a fluorescein-conjugated anti-rabbit IgG (Jackson ImmunoResearch), and nuclei were stained with 4-6-diamidino-2-phenylindole-2HCl (DAPI, Sigma). Coverslips were examined by laser confocal microscopy (Nikon). For quantification, 250 cells per coverslip were counted.
Infection and Oil Red O Staining
Packaging of the retroviral particles was achieved by transfecting the LXSN expression plasmids into EcoPack-293 packaging cell line (Clontech) with the Fugene 6 reagent (Roche) according to the protocol. Forty-eight hours after transfection, supernatants from packaging cells were collected and filtered through sterile 0.45-μm syringe filters. Twenty-four hours before infection, 3T3F442A mouse preadipocyte cells (Ecacc) were seeded at a density of 2x105 per 60-mm plates. For infection, recipient cells were incubated with viral supernatant containing a final concentration of 4 μg/mL Polybrene (Sigma). After 5 hours, cells were fed with fresh Dulbecco’s modified Eagle’s medium supplemented with 10% newborn calf serum and 100 U/mL penicillin/streptomycin (Gibco) and allowed to grow to 80% confluence. Once confluent cells were differentiated, they were stained with oil red O solution.15 Cells were kept in the presence of the appropriate antibiotic throughout the experiments to maintain stable expression levels of GATA2, GATA2S401A, and GATA2S401D.
RNA Isolation, Reverse-Transcriptase Polymerase Chain Reaction, and Northern Blotting
Total RNA was isolated with TRIzol reagent (Life Technologies) as described by the manufacturer. For reverse-transcriptase polymerase chain reaction (RT-PCR), 1 μg total RNA was reverse-transcribed into cDNA with 1 U/mL of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen) at 42°C for 45 minutes. Primer sequences are available on request. Northern hybridization was performed according to standard techniques.15
Cytokines Antibody Array
Protein extracts (250 μg) from GATA2 mutant–infected 3T3-F442A preadipocytes were incubated with the mouse cytokine antibody array membranes according to the protocol (Panomics, Inc). The active cytokines were visualized by chemiluminescence.
Phagocytosis Studies
Phagocytosis assay was performed by incubating cells with fluorescently labeled zymosan A (S cerevisiae) bioparticles that were fluorescein conjugated (Molecular Probes).3 Nuclei were stained with DAPI 2 μg/mL in PBS buffer. Phagocytic activity was identified with a confocal microscopy (Nikon) and reported as percentage of phagocytizing cells.
Flow Cytometry Analysis
For direct immunofluorescence by flow cytometry, nonconfluent (day –1) 3T3-F442A was analyzed for the expression of F4/80 with F4/80-PE antibody (Caltag Laboratories Inc) as described.5
Subcutaneous Implantation of Preadipocytes, Excision of Fat Pads, and Histology
Infected 3T3-F442A preadipocytes were injected subcutaneously (3x107 cells per site) into the back of athymic mice (Harlan). Seven weeks later, the resulting tissue was dissected and analyzed as described.24 Total RNA was isolated by use of the TRIzol reagent from a portion of dissected tissue and analyzed by RT-PCR as described above.
Statistical Analysis
Data are expressed as mean±SD. Statistical analyses were performed with ANOVA or Student t test as indicated.
Results
Akt Phosphorylates GATA2 In Vitro at Ser401
To analyze the interaction between Akt and GATA2, we first determined whether GATA2 could be phosphorylated by Akt in vitro. We generated HA epitope-tagged vectors containing either a GATA2 wild-type or a nonphosphorylable form in which the putative Akt phosphorylation site at Ser401 was replaced with a nonphosphorylable alanine residue (GATA2S401A). HEK293 cells were transfected with HA-GATA2 wild-type or HA-GATA2S401A, and the 2 variants of GATA2 were immunoprecipitated with an anti–HA-Tag antibody. The immunoprecipitates were combined with a constitutively active form of Akt (caAkt) in the presence of [-32P]ATP to perform an in vitro kinase assay and separated by SDS-PAGE. Autoradiograph showed that caAkt was able to phosphorylate wild-type GATA2 but not GATA2S401A (Figure 1A, top). In contrast, an inactive form of Akt (dnAkt) was not able to phosphorylate HA-GATA2 wild-type or HA-GATA2S401A, indicating that Ser401 of GATA2 is likely to be an Akt target (Figure 1A, top). To confirm that Ser401 is an Akt phosphorylation site in vitro, we analyzed the same membrane by Western blot with anti–Akt-phospho substrate Ab, which specifically recognizes amino acid sequences containing a serine or threonine residue phosphorylated by Akt. We confirmed that only wild-type GATA2 is phosphorylated by caAkt (Figure 1A, middle). These results raise the possibility that GATA2 may be a novel substrate for Akt.
Phosphorylation of GATA2 at Ser401 in Response to Insulin
Next, we tested the hypothesis that insulin stimulates GATA2 Ser401 phosphorylation in living cells. For this experiment, we used HEK293 cells, which are an optimal model for transfection assays. Cells transfected with HA-GATA2, HA-GATA2S401A, or HA empty vector were labeled with [32P]orthophosphate and stimulated with insulin for 5, 15, or 30 minutes. Thereafter, GATA2 was immunoprecipitated with an anti–HA-Tag Ab and separated by SDS-PAGE. We found that insulin induced a significant phosphorylation of GATA2 after 30 minutes of treatment, whereas in GATA2S401A-transfected cells, we did not observe any increase in 32P content (Figure 1B). These data indicate that GATA2 is phosphorylated at Ser401 in an insulin-dependent manner. We next investigated whether GATA2 phosphorylation is regulated by the insulin/IR/Akt signaling pathway. To increase activation of the insulin/IR/Akt signaling pathway, cells were cotransfected with either HA-GATA2 or HA-GATA2S401A and IR vectors and stimulated with insulin. GATA2 and GATA2S401A were immunoprecipitated and separated by SDS-PAGE, and phosphorylation levels were detected by Western blotting with an anti–Akt-phospho substrate antibody. Phosphorylation was already induced after 10 minutes in GATA2-transfected cells, but not in cells transfected with GATA2S401A (Figure 1C), indicating that insulin-dependent phosphorylation of Ser401 is activated by Akt in an insulin receptor dependent manner.
Phosphorylation of GATA2 at Ser401 in Response to Insulin Is Inhibited by Wortmannin
To determine whether insulin-dependent phosphorylation of GATA2 is directly mediated by the IR/PI3K/Akt signaling pathway, we performed phosphorylation experiments in the presence of wortmannin, an inhibitor of PI 3-kinase activity. For these experiments, HEK293 cells were cotransfected with either HA-GATA2 or HA-GATA2S401A and IR vectors. Pretreatment of cells with wortmannin abolished the ability of insulin to stimulate phosphorylation of GATA2 (Figure 1D). Because it was previously shown that GATA factors might be phosphorylated by mTOR and MAPK, we investigated whether these pathways could affect Ser401 phosphorylation.25,26 Neither the mTOR inhibitor rapamycin (Figure 1D) nor the MAPK inhibitor PD098059 (data not shown) affect insulin-dependent GATA2 phosphorylation at Ser401. These results further support a role for Akt in the phosphorylation of GATA2 at Ser401.
Role of Ser401 in Regulating Function of GATA2
Next, we tested the hypothesis that insulin impairs the transactivation potential of GATA2 through phosphorylation at Ser401 by Akt. We analyzed the effect of insulin on GATA2 interaction with specific DNA sequences by an EMSA assay. We found that insulin reduced the GATA2-DNA complex in HEK293 cells transfected with HA-GATA2 (Figure 2A). Immunoblotting with the anti–HA-Tag antibody performed on the same extracts showed that insulin reduced the nuclear amount of GATA2 protein (Figure 2B).
To determine whether phosphorylation at Ser401 is involved in the regulation of the binding activity and nuclear protein amount, we analyzed the effects of insulin in cells transfected either with GATA2S401A or with a mutant in which Ser401 was replaced with aspartate (GATA2S401D), which mimics a constitutive phosphorylation state. We found that GATA2S401A was bound to the DNA sequences in either presence or absence of insulin. Instead, the complex formation was inhibited in cells transfected with the HA-GATA2S401D vector (Figure 2C). A Western blot assay performed with nuclear extracts from the same cells showed that the nuclear protein expression of GATA2S401D was dramatically reduced with respect to GATA2S401A (Figure 2D). These results suggest that phosphorylation of GATA2 at Ser401 modulates its intracellular localization.
Subcellular Localization of GATA2 Mutants
Then, we asked whether phosphorylation of Ser401 affects the subcellular localization of GATA2. For this experiment, we used SAOS-1 cells because they are easily transfectable and, having a good nucleus to cytoplasm proportion, they allow optimal visualization of cytoplasm/nucleus trafficking. We transiently transfected these cells with either HA-GATA2S401A or HA-GATA2S401D and performed confocal immunofluorescence analysis with an anti–HA-Tag antibody. We observed that GATA2S401A was localized to both nucleus and cytoplasm, whereas GATA2S401D was prevalently cytoplasmatic (Figure 2E). Taken together, these results suggest that the insulin-induced phosphorylation at Ser401 modulates GATA2 DNA binding activity because it reduces the nuclear amount of the protein.
Role of GATA2 Ser401 in Adipocyte Differentiation In Vitro
GATA2 is involved in the regulation of the preadipocyte-adipocyte differentiation. Thus, to investigate the physiological role of Ser401 phosphorylation in preadipocyte-adipocyte differentiation, we took advantage of 3T3-F442A preadipocyte cells, a widely used culture model for adipogenesis. Because the differentiation process is extended for 12 to 14 days in 3T3-F442A, we elected to use retroviral constructs to introduce and maintain GATA2 mutant expression in these cells. Retrovirus-infected preadipocytes are known to retain a good level of expression through differentiation.10 Therefore, we generated retroviral constructs encoding GATA2 (LX-GATA2), GATA2S401A (LX-GATA2S401A), and GATA2S401D (LX-GATA2S401D) and used them to produce various 3T3-F442A clones. We also infected 3T3-F442A with the empty vector (LXSN, mock) for control. We confirmed by RT-PCR that expression of GATA2 wild-type and mutant constructs was maintained during differentiation for up to 16 days (Figure 3A). Subsequently, we analyzed the ability of the infected clones to differentiate. Adipogenesis was normal in 3T3-F442A mock-infected cells. In contrast, both GATA2 and GATA2S401A exerted an inhibitory effect on intracellular lipid accumulation, whereas 3T3-F442A infected with LX-GATA2S401D exhibited normal adipogenesis, as assessed by oil red O staining at day 16 (Figure 3B). Northern blot analysis showed a marked decreased in PPAR- expression in 3T3-F442A infected with LX-GATA2 and LX-GATA2S401A compared with LX-GATA2S401D– and mock-infected 3T3-F442A cells (Figure 3C). Thus, phosphorylation of GATA2 on Ser401 appears to be crucial for the adipogenesis program in vivo.
Role of GATA2 Ser401 in Phagocytic Activity
Preadipocytes exhibit phagocytic activity similarly to macrophages.3 An inverse relationship between adipose cell and macrophage differentiation has been described.3 To determine whether the phosphorylation of GATA2 on Ser401 could affect common functions between preadipocytes and macrophages, we measured phagocytic activity in growing preadipocytes infected with GATA2 mutants. LX-GATA2– and LX-GATA2S401A–infected cells showed a significantly increased phagocytosis with respect to the LX-GATA2S401D and control cells (Figure 4A), indicating that the phosphorylation of GATA2 on Ser401 is involved in the link between adipose tissue and immunity processes.
Many mediators of inflammation are secreted by adipocytes. To investigate whether the 2 mutants, GATA2S401A and GATA2S401D, display a different profile of expression of inflammatory proteins, we used a cytokine protein array. Cell lysates from LX-GATA2S401A– or LX-GATA2S401D–infected 3T3F442A preadipocytes were incubated with arrays containing antibodies directed against various inflammatory proteins. We found that cells infected with LX-GATA2S401A express significantly higher levels of macrophagelike cytokines such as monocyte chemotactic protein-1 (MCP-1), thrombopoietin (TPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 4 (IL-4) compared with cells infected with LX-GATA2S401D (Figure 4B). We confirmed the upregulation of MCP-1 in GATA2S401A with respect to GATA2S401D by RT-PCR (Figure 4C). In contrast, analysis by both RT-PCR (Figure 4C) and flow cytometry (data not shown) of F4/80, a marker for mature macrophages, was negative in 3T3-F442A cells infected with LXSN, LX-GATA2, LX-GATA2S401A, and LX-GATA2S401D (Figure 4C) and in differentiated 3T3-F442A adipocytes (data not shown).
Role of GATA2 Ser401 in Adipocyte Differentiation In Vivo
Next, we performed experiments to assess the role of GATA2 phosphorylation at Ser401 in animal models. Implanted 3T3-F442A preadipocytes injected into nude mice fed a high-fat diet gave rise to fat pads indistinguishable from normal adipose tissue.24 To examine whether the effects of Ser401 phosphorylation are maintained in vivo, we subcutaneously injected into athymic mice 3T3-F442A preadipocytes infected with the different LX-GATA2 constructs or LXSN. Mice were fed a high-fat diet to trigger adipocyte differentiation. Seven weeks later, we dissected the resulting tissue. We found that preadipocytes infected with LXSN and LX-GATA2S401D were able to differentiate into adipocytes, whereas cells bearing LX-GATA2 and LX-GATA2S401A formed pads composed mostly of undifferentiated fibroblastlike cells and rare adipocytes (Figure 5A). To confirm that the tissues were derived from the injected preadipocytes, we assessed hGATA2 expression by RT-PCR (Figure 5B). All fat pads were positive for hGATA2 expression. The area covered by adipocytes in the LX-GATA2S401D fat pad was 5-fold higher than LX-GATA2S401A (P<0.01; data not shown). Moreover, LX-GATA2S401D–infected cells formed both unilocular and multilocular adipocytes, which were not observed in LX-GATA2S401A fat pads.
Because we found increased expression of MCP-1 in cultured preadipocytes infected with retrovirus encoding GATA2 and GATA2S401A, we evaluated mRNA expression of mouse MCP-1 and F4/80 in fat pads (Figure 5B). Both MCP-1 and F4/80 mRNA were upregulated in fat pads derived from implants of 3T3-F442A cells infected with GATA2 and GATA2S401A (Figure 5B).
Discussion
Obesity-related inflammation is emerging as a cause of systemic insulin resistance and atherosclerosis. Inflammation plays a major role in the development of cardiovascular events. In fact, advanced or unstable atherosclerotic plaques are known to be in an even higher state of inflammation than stable plaques. Thus, obesity has been hypothesized to be a proinflammatory state, increasing the vulnerability of atherosclerotic plaques. However, mechanisms explaining how adiposity triggers inflammation are still elusive.27 It is recognized that adipose and hematopoietic tissues share some characteristics. In fact, some in vitro studies showed that preadipocytes could behave like or even transform into macrophages.3,4 More recently, a specific accumulation of macrophages in adipose tissue has been demonstrated. Inflammatory markers would derive only from these macrophages, which would be responsible for the effects on insulin sensitivity and vascular homeostasis.5,6 However, it remains unclear which factors balance the preadipocyte fate toward an adipocyte or macrophage phenotype, as well as which signals drive monocyte/macrophage accumulation into adipose tissue.
Adipose tissue function is controlled by hormonal cues that, through signals delivered to the nucleus, modulate the transcription of enzymes and molecules that regulate lipid metabolism.11
Our results show that GATA2 is a novel Akt-regulated transcription factor involved in adipogenesis. In addition, our findings suggest that the Akt/GATA2 axis could act as a balance between the adipogenic and the macrophagelike phenotype of preadipocytes. GATA2 when phosphorylated by Akt is excluded from the nucleus, and preadipocytes differentiate, losing their ability to exert inflammatory functions. In contrast, when GATA2 is inadequately phosphorylated, it blocks adipogenesis by inhibiting PPAR- expression, thus maintaining preadipocytes in a macrophagelike state. In fact, preadipocytes expressing wild-type GATA2 and GATA2S401A show increased phagocytic behavior compared with preadipocytes bearing GATA2S401D. This is consistent with previous observations suggesting that GATA2 plays a major role in phagocytosis. In fact, macrophages infected with P carinii lose GATA2 expression and consequently phagocytic activity. Furthermore, once GATA2 is overexpressed in infected macrophages, they reacquire phagocytic activity.28 Therefore, our results corroborate the observation that preadipocytes behave like macrophages.3 However, we observed that preadipocytes expressing GATA2 do not bear a typical macrophage marker like F4/80, indicating that they are not fully converted into macrophages. Our data support the hypothesis that GATA2 activity in these preadipocytes takes part in the complex regulation of monocyte/macrophage trafficking, generating specific signals necessary to drive monocyte accumulation among adipocytes.9 In fact, we show that in preadipocytes an unchecked GATA2 (GATA2S401A) leads to increased MCP-1 and GM-CSF expression, while the opposite is seen when GATA2 activity is inhibited (GATA2S401D). Noteworthy, both MCP-1 and GM-CSF play a role in monocyte diapedesis, although at different extents.29,30 Our results are compatible with recent observations suggesting that the inflammatory burden of adiposity is due to macrophage cell accumulation in the adipose tissue.6,9 Moreover, because GATA2 is involved in vascular cell adhesion molecule-1 expression, an adhesion molecule determinant for monocyte adhesion to other cells,31 it is plausible that GATA2 action in endothelial cells could facilitate monocyte diapedesis in the vessel wall, accounting also for a possible proatherosclerotic effect of this transcription factor.
To determine the role of GATA2 on adipogenesis in vivo, we used the fat pad formation model. In mice injected with mock-infected or GATA2S401D-infected cells, adipocytes develop normally. In contrast, in mice injected with preadipocytes bearing an unchecked GATA2, fat pad formation is blocked, and we observed increased mRNA expression of both MCP-1, confirming our in vitro observation, and F4/80. Because F4/80 was not expressed by preadipocytes in vitro, it is conceivable that it derives from monocytes that migrated near injected cells in response to the increased MCP-1 expression.
We can speculate that insulin triggers adipogenesis to maintain metabolic homeostasis by restraining in preadipocytes both repressors of metabolic program like FOXO-1, as well as factors regulating cell cycle and inflammatory properties like GATA2. Therefore, in insulin resistance, unchecked FOXO1 might inhibit energy storage, causing metabolic derangement.15 Simultaneously, unchecked GATA2 might facilitate monocyte homing and macrophage differentiation via an increased expression of factors such as MCP-1, GM-CSF, and IL-4 from preadipocytes.29–32 Interestingly, a synthetic inhibitor for GATA2 has recently been developed and shown to act as an antiinflammatory agent.33
Our data suggest that insulin regulating GATA2 can direct preadipocytes toward an adipogenic program, whereas insulin resistance, resulting from either genetic or metabolic factors, would facilitate a GATA2-sustained inflammatory program. Therefore, the inhibition of GATA2 could be investigated as a new strategy to lower obesity-related inflammation to prevent the development of late complications of obesity such as diabetes and plaque rupture.
Acknowledgments
This study was supported by grants from the Italian Ministry of University (PRIN 2003067733-001 to Dr Lauro; PRIN 2003067733-006 to Dr Federici), Italian Ministry of Health (RFS 2001, 2002, and 2003 to Dr Federici), and a grant to University of Tor Vergata Center of Excellence on Genomic Risk Assessment (MIUR CE00277884 to Dr Lauro). Drs Menghini and Hribal are supported by a grant from the University of Tor Vergata.
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