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Induce Expression of the Forkhead Transcription Factor Gene Foxc2 in 3T3-L1 Adipocytes via PI3K and ERK 1/2-Dependent Pathways
Department of Medical Biochemistry (L.M.G., K.T.), Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway; Medical Genetics (A.C., S.E.), Department of Medical Biochemistry, Göteborg University, SE-405 30 Göteborg, Sweden; and Department of Biochemistry (N.M.), Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan
Address all correspondence and requests for reprints to: Kjetil Taskén, M.D., Ph.D., Department of Medical Biochemistry, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1112, Blindern, N-0317 Oslo, Norway. E-mail: kjetil.tasken{at}basalmed.uio.no.
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ABSTRACT |
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and insulin induce Foxc2 mRNA in differentiated 3T3-L1 cells with the kinetics of an immediate early response (1–2 h with 100 ng/ml insulin or 5 ng/ml TNF). This induction is, in both cases, attenuated by the PI3K inhibitor wortmannin as well as the MAPK kinase inhibitor PD98059. Furthermore, we show that stimulation of 3T3-L1 adipocytes with phorbol-12-myristate-13-acetate or 8-(4-chlorophenyl)thio-cAMP induces the expression of Foxc2. Interestingly, we find that the basal level of Foxc2 mRNA is down-regulated whereas hormonal responsiveness increases during differentiation of 3T3-L1 from preadipocytes to adipocytes. At the protein level, immunoblots with Foxc2 antibody demonstrated an induction of Foxc2 by insulin and TNF in nuclear extracts of 3T3-L1 adipocytes. EMSA of nuclear proteins from phorbol-12-myristate-13-acetate- and TNF-treated 3T3-L1 adipocytes using a forkhead consensus oligonucleotide revealed specific binding of a Foxc2/DNA complex. In conclusion, our data suggest that insulin and TNF regulate the expression of Foxc2 via a PI3K- and ERK 1/2-dependent pathway in 3T3-L1 adipocytes. Also, signaling pathways downstream of PKA and PKC induce the expression of Foxc2 mRNA. |
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subunit of PKA that lowers the threshold for activation by cAMP, increases perturbation of the PKA signaling pathway, which in turn induces the levels and activity of hormone-sensitive lipase (HSL) and UCP1. HSL metabolizes triglycerides to FFAs, and UCP1 has the ability to dissipate energy through uncoupling of oxidative phosphorylation, which will produce heat instead of generating ATP. According to this model, the energy content of FFAs, released by HSL, will be dissipated through the induction of UCP1 in response to ß-adrenergic stimuli. Thus, these mice display a lean phenotype with lowered plasma levels of FFAs, glucose, and insulin and increased oxygen consumption (1). Furthermore, Foxc2 mRNA is up-regulated in wild-type mice fed a high-fat diet as compared with standard diet, indicating that Foxc2 is regulated in response to diet energy content. After elevated Foxc2 levels, the metabolic rate is increased in the sense that excess calories will have an increased tendency to dissipate heat rather than being stored as triglyceride droplets. Taken together, these findings suggest that Foxc2 is an important regulator of energy homeostasis.
TNF is a proinflammatory cytokine produced systemically by macrophages and lymphocytes after inflammatory stimulation or trauma and increases rapidly during experimental injury induced by cerebral ischemic, excitotoxic, and traumatic injury (2). In some chronic diseases, TNF promotes the syndrome of wasting and malnutrition known as cachexia (3, 4). TNF has also been implicated as an important modulator of energy metabolism, particularly in adipocytes (5, 6). Adipose tissue produces TNF, and elevated levels of TNF are associated with obesity and non-insulin-dependent diabetes mellitus (5, 6, 7). Furthermore, chronic treatment of 3T3-L1 adipocytes with TNF inhibits glucose uptake and induces a moderate decrease of insulin-stimulated phosphorylation of the insulin receptor and inhibition of insulin-promoted tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) (8, 9). Acute treatment with TNF, however, induces tyrosine phosphorylation of IRS-1 and its interaction with PI3K, eliciting growth factor- or insulin-like effects (10, 11). Furthermore, both insulin and TNF have been shown to signal through ERK 1/2 (also called p44/p42 mitogen activated protein kinases) in 3T3-L1 adipocytes (12, 13).
The present study was performed to investigate the hormonal regulation of Foxc2 in 3T3-L1 adipocytes. We find that insulin and TNF induce the expression of Foxc2 via a PI3K- and ERK 1/2-dependent mechanism. Moreover, we find that phorbol-12-myristate-13-acetate (TPA) induces the Foxc2 mRNA level through perturbation of the PKC signaling pathway. Lastly, we observe that treatment with a cell-permeable cAMP analog induces Foxc2, indicating regulation also by a ß-adrenergic pathway involving PKA. To maintain energy homeostasis, we postulate that Foxc2 in adipocytes is transiently induced by elevated levels of insulin and TNF produced in response to high-fat diet or trauma.
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shows a representative Northern blot of Foxc2 from untreated differentiated 3T3-L1 cells or cells treated with TPA (100 nM) for 2–24 h. Maximal levels of Foxc2 mRNA were observed after 2 h of stimulation, and the induction was sustained for up to 12 h (Fig. 1, A and C). After 24 h of stimulation with TPA, Foxc2 mRNA was back to basal levels. Because of the early and strong TPA-mediated induction of Foxc2, we performed a shorter time course where we stimulated 3T3-L1 adipocytes with TPA for 30 min to 2 h (Fig. 1B). TPA induced Foxc2 mRNA weakly after 30 min of stimulation, and maximal levels were observed after 1–2 h of stimulation (Fig. 1C). Basal levels of Foxc2 mRNA also increased during the first 2 h in culture. In contrast to TPA, 4-phorbol (100 nM) did not induce the level of Foxc2 mRNA (not shown). A concentration-dependent increase in the level of Foxc2 mRNA was observed (Fig. 1D) with a half-maximal concentration for induction of Foxc2 of approximately 3 nM TPA and effective concentrations also below 1 nM (not shown).
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in 3T3-L1 Adipocytes, and TNF, according to our previous report, up-regulates Foxc2 steady state mRNA levels (1), we next investigated the regulation of Foxc2 by TNF. Figure 2A shows Foxc2 mRNA from untreated differentiated 3T3-L1 cells or cells treated with either murine or human recombinant TNF (50 ng/ml). Whereas mouse TNF utilizes both type 1 and 2 TNF receptors, human TNF signals only through the type 1 TNF receptor in murine adipocytes. Murine TNF induced Foxc2 mRNA weakly after 1 h of stimulation (not shown) and to maximal levels after 2–6 h of stimulation (Fig. 2, A and B), whereas human TNF was more potent in increasing the level of Foxc2 mRNA (3-fold vs. 2-fold at 2 h of stimulation). After 12 h of stimulation with murine TNF (mTNF), Foxc2 mRNA was back to basal levels. A concentration-response experiment with mTNF showed half-maximal induction of Foxc2 mRNA with 1 ng/ml TNF, and maximal induction was observed with concentrations of TNF above 5 ng/ml (Fig. 2C).
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shows a representative Northern blot of Foxc2 from untreated differentiated 3T3-L1 cells or 3T3-L1 cells treated with insulin (1 µg/ml) in serum-free medium. Maximal levels of Foxc2 were observed after 2–6 h of stimulation (Fig. 3B). A weak induction was observed after 1 h of stimulation (not shown). Increasing concentrations of insulin were added to 3T3-L1 adipocytes in serum-containing medium (Fig. 3C). Induction of Foxc2 mRNA was obtained at a concentration of 100 ng/ml insulin, and maximal induction was observed with 10 µg/ml insulin in all experiments (Fig. 3D). Whereas Foxc2 regulation by insulin and TNF were observed both in cells cultured in the absence and presence of serum, the basal level of Foxc2 were approximately 2-fold higher in the presence of serum, suggesting that serum (growth factors) also induce the level of Foxc2 mRNA. In line with this notion, stimulation of 3T3-L1 adipocytes for 2 h with low levels of IGF-I (100 ng/ml) or insulin (100 ng/ml) in the absence of serum was able to induce the level of Foxc2 mRNA (not shown).
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have been shown to signal through IRS-1 and PI3K in adipocytes (10, 11), we preincubated differentiated 3T3-L1 cells with the PI3K inhibitor, wortmannin, before administration of hormones. Figure 4A shows Northern blot analysis of untreated, differentiated 3T3-L1 cells or cells treated with insulin (10 µg/ml) or human recombinant TNF (50 ng/ml) for 2 h alone or in combination with wortmannin (100 nM) (added 45 min before hormones). Wortmannin clearly attenuated the insulin-, and TNF-mediated induction of Foxc2 mRNA (Fig. 4, A and C). In contrast, wortmannin did not inhibit TPA-mediated induction of Foxc2 (Fig. 4, B and C), indicating that PKC activation is independent of or downstream of PI3K. Similar results were obtained with another PI3K inhibitor, LY294002 (data not shown). Furthermore, both inhibitors had more profound effect on TNF-regulated Foxc2 levels than insulin-regulated Foxc2 levels.
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-stimulated 3T3-L1 adipocytes to a 32P-labeled forkhead oligonucleotide was examined (Fig. 5A). Because Freac 3/Foxc1 (not expressed in adipocytes) and Foxc2 have identical DNA-binding domains, we used the forkhead-binding site characterized for Freac 3 to detect Foxc2 by EMSA (14). Binding to the labeled forkhead consensus site was induced by TPA and TNF (lane 2 vs. lanes 5 and 8). Proteins binding to the labeled DNA fragment could only be displaced by the homologous unlabeled probe (lanes 3 and 6) and not by a mutated oligo (lanes 4 and 7). We further investigated the expression of immunoreactive Foxc2 in nuclear extracts of 3T3-L1 adipocytes (Fig. 5B). Immunoblots with Foxc2 antibody demonstrated detectable levels of Foxc2 protein (62 kDa) under basal conditions (6 h after change of medium). Treatment with recombinant human TNF (50 ng/ml), insulin (10 µg/ml), or TPA (100 nM) for 6 h induced nuclear Foxc2. The level of Foxc2 present in the postnuclear supernatant was low and similar between untreated and stimulated cells (not shown). The observation of an additional weaker band migrating at approximately 66 kDa is similar to what has been reported earlier (15, 16).
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, PPAR
, and sterol-regulatory element binding protein 1 (1). For this reason we wanted to investigate whether the level of Foxc2 mRNA would change during in vitro differentiation of 3T3-L1 cells. The Northern blot in Fig. 6A shows that Foxc2 mRNA is present in 3T3-L1 preadipocytes (harvested 2 d before confluence and 24 h after the last medium change), whereas it is down-regulated at confluence. To address whether 3T3-L1 preadipocytes (before confluence) responded to hormonal stimuli, we treated preconfluent 3T3-L1 cells with insulin (10 µg/ml), recombinant human TNF (50 ng/ml), or TPA (100 nM) for 2 and 6 h (Fig. 6B). The levels of Foxc2 mRNA were not responsive to stimuli by TNF, and insulin-treated cells showed only a weak and transient induction of Foxc2, whereas TPA induced the expression of Foxc2 to a lesser extent than in 3T3-L1 adipocytes (3-fold vs. 
8-fold in differentiated cells). We conclude that basal Foxc2 levels are higher, and hormonal responsiveness lower, in preadipocytes vs. adipocytes.
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-subunit of PKA, rendering these mice more sensitive to ß-adrenergic stimuli (1). In line with this notion, we further showed that upon stimulation with ß-adrenergic agonists, the cAMP level is high and sustained in these mice as compared with ß-agonist-treated wild-type littermates. Because of these findings, we investigated whether cAMP conversely would regulate the expression of Foxc2. Treatment of 3T3-L1 adipocytes with 8-CPTcAMP (100 µM) induced Foxc2 mRNA with rapid and transient kinetics and with maximal induction after 2 h of stimulation (Fig. 7A). At earlier time points (not shown), we observed a 2-fold induction of Foxc2 mRNA after 30 min of stimulation with the cAMP analog. In a concentration-response experiment depicted in Fig. 7B, we show that induction of Foxc2 mRNA by 8-CPTcAMP was observed at 10 µM, and maximal observed induction of Foxc2 expression was obtained with 400 µM 8-CPTcAMP. Figure 7C shows a Northern blot of 3T3-L1 adipocytes stimulated for 2 h with 8-CPTcAMP (100 µM) alone or in combination with the PKA inhibitor KT5720 (100 nM) (added 45 min before the cAMP analog). KT5720 clearly attenuated the cAMP-mediated induction of Foxc2 mRNA from 3-fold to basal levels. Similar results were obtained with another PKA inhibitor, H89 (data not shown). Figure 7D shows a Northern blot of 3T3-L1 adipocytes stimulated with the cAMP analog for 2 h alone or in combination with wortmannin (100 nM) (added 45 min before 8-CPTcAMP). Wortmannin attenuated the cAMP-mediated induction of Foxc2 mRNA, indicating that cAMP/PKA signaling to Foxc2 in 3T3-L1 adipocytes may involve downstream activation of PI3K.
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have been shown to signal through ERK 1/2 in 3T3-L1 adipocytes (12, 13), and cAMP has been shown to cross-talk to the ERK-pathway in adipocytes (17), we preincubated differentiated 3T3-L1 cells with the MAPK kinase (MEK) inhibitor, PD98059, before stimulation. Figure 8A shows a Northern blot of untreated, differentiated 3T3-L1 cells or cells treated with insulin (10 µg/ml), human recombinant TNF (50 ng/ml), or 8-CPTcAMP (100 µM) for 2 h alone or in combination with PD98059 (50 nM) (preincubated for 45 min). PD98059 clearly attenuated the insulin and TNF-mediated, but not the 8-CPTcAMP-mediated, induction of Foxc2 mRNA. Again, TNF-regulated Foxc2 mRNA levels were more sensitive to inhibition by PD98059 than insulin-regulated Foxc2 levels.
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, insulin, or cAMP, we pretreated 3T3-L1 adipocytes with TPA for 24 h to inhibit PKC before administration of hormones or cAMP analog. Figure 8B
shows a Northern blot of untreated, differentiated 3T3-L1 cells and cells treated with TPA (100 nM), TNF (50 ng/ml), insulin (10 µg/ml), or 8-CPTcAMP (100 µM) for 2 h alone or after a 24-h TPA pretreatment (100 nM). TPA pretreatment abolished the TPA-mediated induction, but not the TNF, insulin, or 8-CPTcAMP-mediated induction of Foxc2 mRNA.
We next investigated whether cAMP/PKA is implicated in the induction of Foxc2 expression by insulin and TNF signal by pretreating 3T3-L1 adipocytes with KT5720. The Northern blot in Fig. 8C shows that KT5720 did not inhibit the insulin- or TNF-mediated induction of Foxc2 mRNA, suggesting that the mechanisms by which PI3K/ERK induce the expression of Foxc2 mRNA are not dependent on PKA.
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DISCUSSION |
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and insulin (5, 7, 18). Here we report that short-term stimulation by insulin and TNF induce the anti-thrifty gene Foxc2 with the kinetics of an immediate early response (1–2 h after stimulation), which in the healthy individual would serve to counteract insulin resistance and obesity. We find that the PI3K inhibitor, wortmannin, as well as the MEK inhibitor PD98059, attenuate the induction of Foxc2 mRNA, indicating that PI3K and ERK 1/2 are activated upon stimulation of insulin- and TNF receptors most likely through tyrosine phosphorylation of the docking protein IRS-1 (10, 11). The early and transient induction of Foxc2 mRNA by TNF and insulin indicate that Foxc2 responds acutely to elevated levels of these regulators. In contrast, upon long-term stimulation with TNF or insulin, which would mimic the situation in obesity and/or insulin resistance, the regulation of Foxc2 is terminated and Foxc2 mRNA is at basal levels. The finding that chronically TNF-stimulated adipocytes display inhibited tyrosine phosphorylation of IRS-1 in response to insulin due to TNF-mediated serine phosphorylation of IRS-1, which disrupts association with PI3K, is in line with this notion (7, 8, 19, 20). We further show that recombinant human TNF is a more potent inducer of Foxc2 expression in murine 3T3-L1 adipocytes than murine TNF. TNF initiates its actions by binding to either of two receptors (21). The extracellular domains of the receptors share homology with one another, but the intracellular domains do not display sequence similarities, and neither receptor contains protein tyrosine kinase activity (21, 22). In contrast to mouse TNF that utilizes both receptors, the human form of TNF signals only through the type 1 TNF receptor in murine adipocytes, which mediates signaling through IRS-1 (10, 23), and indicates that the type 1 TNF receptor-IRS-PI3K-ERK 1/2 pathway is responsible for regulation of Foxc2.
We further show that the phorbol ester TPA induces the level of Foxc2 mRNA with the kinetics of an immediate early response in 3T3-L1 adipocytes. However, treatment with wortmannin does not attenuate the TPA-mediated induction of Foxc2 and, conversely, chronic TPA treatment to down-regulate PKC does not abrogate TNF- and insulin-regulated Foxc2 expression. These observations suggest that separate pathways are involved in the regulation of Foxc2 by TNF/insulin/PI3K/ERK 1/2 and TPA. PI3K 3-phosphorylates inositide lipids, which are then able to bind to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase and protein kinase B (PKB) and thus regulates the phosphorylation and activation of PKB by phosphoinositide-dependent protein kinase. Furthermore, phosphatidylinositol 3,4,5-trisphosphate (PIP3), the major product formed by active PI3K, can bind to and activate a number of atypical PKC isoforms (reviewed in Ref. 24). TPA, however, does not activate the atypical PKCs. A phorbol ester lacking the ability to activate PKC, 4-phorbol, did not induce the level of Foxc2 mRNA (not shown), suggesting that TPA regulation involves activation of classical PKCs. Such classical PKCs are expressed in 3T3-L1 cells, even though levels are lower than in preadipocytes (25, 26). Stimulation of 3T3-L1 adipocytes with TPA has been shown to increase the levels of PIP3 without activation of PI3K (27) and because wortmannin did not block the effect of TPA on Foxc2 in our study, this suggests that the effects of insulin/TNF and classical PKC may converge downstream of PI3K at the level of PIP3 (Fig. 9).
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did not, and insulin did only weakly and transiently, induce the expression of Foxc2 mRNA. This is consistent with low levels of insulin receptors expressed on undifferentiated 3T3-L1 cells (31). At the mRNA level, there are no significant changes in TNFR1 and TNFR2 expression between immature vs. mature 3T3-L1 adipocytes (23). However, the expression levels of IRS-1, p110ß/PI3K, and PKBß have been reported to increase during adipocyte differentiation (32, 33, 34). This up-regulation places Foxc2 under hormonal control with lowered, but inducible, levels in the mature adipocyte. This may serve to terminate the embryonal function of this winged helix/forkhead transcription factor and switch to its adult function as a hormonally controlled regulator of metabolic efficiency.
We further show that 8-CPTcAMP induces the level of Foxc2 with rapid and transient kinetics. In addition, the PKA inhibitor KT5720 inhibited the cAMP-mediated, but not the insulin- and TNF-mediated, induction of Foxc2 mRNA, suggesting that PKA is involved in regulating the level of Foxc2 by stimuli other than insulin and TNF (Fig. 9). Treatment with wortmannin attenuated the cAMP-mediated induction of Foxc2 expression, and forskolin and insulin had additive effects on the induction of Foxc2 mRNA (not shown), suggesting the involvement of separate downstream targets for PKA and PI3K and/or that PI3K is activated by PKA. PKA has been shown to phosphorylate the p85-regulatory subunit of PI3K stimulating the formation of a PI3K-p21 Ras complex in TSH-stimulated thyroid cells (35). So far, we have been unable to show induction of Foxc2 expression after administration of ß-adrenergic agonists in 3T3-L1 adipocytes, but we have recently shown that ß-adrenergic stimulation of adipocytes from Foxc2 transgenic mice resulted in rapid induction of cAMP levels (1).
This study has demonstrated that Foxc2 expression in adipocytes is induced by insulin and TNF via activation of PI3K and ERK 1/2. Furthermore, PKC and PKA also induce expression of Foxc2 mRNA. We further show that Foxc2 is expressed in preadipocytes and that it is down-regulated during differentiation, suggesting that Foxc2 is an early marker for adipocyte differentiation. In line with this notion, we have previously shown that Foxc2 up-regulates the expression of CCAAT/enhancer binding protein-, PPAR, and sterol-regulatory element binding protein 1, which are important transcription factors in the differentiation program (1). To our knowledge, Foxc2 is the only known gene that can counteract most, if not all, of the symptoms associated with obesity: hypertriglyceridemia, insulin resistance, and, most likely, the associated clinical syndrome of type 2 diabetes. Indeed, FOXC2 levels are correlated with insulin sensitivity in humans (Klannemark, M., E. Carlsson, A. Cederberg, C. Kösters, H. Tornquist, H. Storgaard, A. Vaag, L. Groop, S. Enerbäck, and M. Ridderstråle, unpublished data). Taken together, hormonal regulation of Foxc2 expression in adipocytes seems to be a key event in the maintenance of energy homeostasis and in regulation of metabolic efficiency.
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MATERIALS AND METHODS |
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(R&D Systems, Minneapolis, MN) was dissolved in PBS containing 0.1% fat-free BSA (Sigma, St. Louis, MO). Insulin (Sigma) was dissolved in distilled water, pH 4.5. 8-CPTcAMP (Sigma) was dissolved in distilled water. TPA and 4-phorbol (Sigma) were dissolved in 96% EtOH. The PKA inhibitor KT5720 (Calbiochem, San Diego, CA), the PI3K inhibitor wortmannin (Calbiochem), and the MEK inhibitor PD98059 (Calbiochem) were dissolved in dimethyl sulfoxide. Isobutylmethylxanthine was dissolved in 40 mM NaOH and dexamethasone was dissolved in distilled water. All stock solutions were stored at -20 C.
Cell Culture and Differentiation
Murine 3T3-L1 fibroblasts (American Type Culture Collection, Manassas, VA) were grown in DMEM containing 4.5 g/liter glucose, 10% heat-inactivated calf serum, 50 U/ml penicillin, 50 µg/ml streptomycin, anti-pleuropneumonia-like organism (PPLO, targets mycoplasma) agent, and maintained in a humidified incubator with 5% CO2 at 37 C. The cells were passaged at approximately 70% confluence. For differentiation experiments, cultures were maintained for 1–2 d at confluence and then switched to differentiation medium (medium supplied with 1 µg/ml insulin, 0.5 mM isobutylmethylxanthine, and 1 µM dexamethasone). The cells were maintained in differentiation medium for 3 d, followed by incubation in medium containing 1 µM insulin for 3 d. After this time, cells were cultivated without insulin, and fresh medium was changed every day. Experiments were conducted in cells at d 11 or 13 (adipocytes) or in cells at approximately 80% confluence (preadipocytes).
RNA Extraction and Northern Blot Analysis
Total RNA from cell cultures and tissue specimens was extracted by the guanidine isothiocyanate/CsCl method as previously described (36, 37). Northern blot analysis was performed using 20 µg total RNA as previously described (38). Mouse cDNA probe for Foxc2 was prepared by PCR using primers directed against 3'-untranslated region of the mouse Foxc2 gene. Foxc2 cDNA was labeled with [-32P]dCTP using megaprime DNA labeling system (Amersham Pharmacia Biotech, Arlington Heights, IL) to a specific activity of 1.0–2.0 x 109 cpm/µg. Hybridization and washing of filters were performed as previously described (38). Northern blots were assessed by ß-scintillation counting using InstantImager (Packard) and subjected to autoradiography using Amersham Pharmacia Biotech Hyperfilm MP. In the figures, bars above Northern blots represent actual cpm over cpm of control subtracted from background given as relative intensity.
Preparation of Nuclear Extracts
3T3-L1 adipocytes (10-cm culture dishes) were scraped in HBSS containing 0.1% fatty acid-free BSA, harvested by centrifugation at 320 x g at 4 C for 5 min, and washed in cold PBS. Cell pellets were resuspended in 450 µl hypotonic buffer (10 mM Tris, pH 7.6, 10 mM NaCl, 3 mM MgCl2) containing the protease inhibitors ALLN (N-acetyl-Leu-Leu-Nle-CHO; 50 µM; Roche, Minneapolis, MN), phenylmethylsulfonyl fluoride (PMSF) (0.5 mM; Roche) and Complete protease inhibitor mix (1 tablet/10 ml; Roche) followed by addition of 0.5% NP-40 (Sigma). The nuclei were pelleted by centrifugation at 130 x g at 4 C for 5 min. The postnuclear supernatant was stored at -70 C until analysis. Nuclei were resuspended in 1 ml hypotonic buffer followed by centrifugation at 130 x g at 4 C for 5 min. Pellets were resuspended in 100 µl of a buffer containing 5 mM HEPES (pH 7.9), 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol. Complete protease inhibitor mix (1 tablet/10 ml), 50 µM ALLN, and 0.5 mM PMSF, 1/10 volume of 4 M NaCl were added, and samples were incubated on a roller for 30 min at 4 C followed by centrifugation at 30,000 x g for 20 min at 4 C. The supernatants (nuclear extracts) were stored at -70 C until analysis.
DNA-Protein Complex Analysis
EMSAs were performed using double-stranded 32P end-labeled forkhead consensus oligonucleotide (5'-GATCCCTTAAGTAAACAGCATGAGATC-3') (14). For each reaction, 5,000 cpm of labeled probe was incubated with 5 µg of crude nuclear proteins from 3T3-L1 adipocytes and 1.0 µg of poly dI:dC in a buffer containing 5 mM HEPES, pH 7.9, 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM PMSF with 100 mM KCl at room temperature for 15 min. Competition experiments were performed in the presence of 250-fold molar excess of unlabeled probe or with a nonspecific forkhead sequence (5'-GATCCAGGCCGTAACAGCATGAGATC-3') (14). Samples were run in 6% nondenaturing polyacrylamide gels at 150 V in Tris-glycine buffer (50 mM Tris, pH 8.5; 380 mM glycine; 2 mM EDTA) at 4 C. Subsequently, gels were dried and subjected to autoradiography.
Immunoblotting
Nuclear extracts from 3T3-L1 cells were diluted in SDS sample buffer and denatured 5 min at 100 C before being subjected to SDS-PAGE (4% stacking gel, 10% separating gel). Forty micrograms of total protein were loaded in each lane, subjected to electrophoresis, and subsequently transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) by electroblotting. The membranes were blocked in a solution containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20, and 5% milk and incubated with a monoclonal antibody against human Foxc2 (15) in blocking solution. Membranes were washed in a solution containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20. Immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech) using a horseradish peroxidase-conjugated secondary antibody (1:20,000) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: 8-CPTcAMP, 8-(4-Chlorophenyl)thio-cAMP; HSL, hormone-sensitive lipase; IRS, insulin receptor substrate; MEK, MAPK kinase; mTNF, murine TNF; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PKB, protein kinase B; PMSF, phenylmethylsulfonyl fluoride; TPA, phorbol-12-myristate-13-acetate; UCP1, uncoupling protein 1; WAT, white adipose tissue.
Received for publication August 13, 2001. Accepted for publication December 3, 2001.
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 G. B. Di Gregorio, R. Westergren, S. Enerback, T. Lu, and P. A. Kern Expression of FOXC2 in adipose and muscle and its association with whole body insulin sensitivity Am J Physiol Endocrinol Metab, October 1, 2004; 287(4): E799 - E803. [Abstract] [Full Text] [PDF]
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 D. Ait-Ali, V. Turquier, L. Grumolato, L. Yon, M. Jourdain, D. Alexandre, L. E. Eiden, H. Vaudry, and Y. Anouar The Proinflammatory Cytokines Tumor Necrosis Factor-{alpha} and Interleukin-1 Stimulate Neuropeptide Gene Transcription and Secretion in Adrenochromaffin Cells via Activation of Extracellularly Regulated Kinase 1/2 and p38 Protein Kinases, and Activator Protein-1 Transcription Factors Mol. Endocrinol., July 1, 2004; 18(7): 1721 - 1739. [Abstract] [Full Text] [PDF]
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 A. Green, J. M. Rumberger, C. A. Stuart, and M. S. Ruhoff Stimulation of Lipolysis by Tumor Necrosis Factor-{alpha} in 3T3-L1 Adipocytes Is Glucose Dependent: Implications for Long-Term Regulation of Lipolysis Diabetes, January 1, 2004; 53(1): 74 - 81. [Abstract] [Full Text] [PDF]
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 K. TASKEN and E. M. AANDAHL Localized Effects of cAMP Mediated by Distinct Routes of Protein Kinase A Physiol Rev, January 1, 2004; 84(1): 137 - 167. [Abstract] [Full Text] [PDF]
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 B. M. Kriederman, T. L. Myloyde, M. H. Witte, S. L. Dagenais, C. L. Witte, M. Rennels, M. J. Bernas, M. T. Lynch, R. P. Erickson, M. S. Caulder, et al. FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome Hum. Mol. Genet., May 15, 2003; 12(10): 1179 - 1185. [Abstract] [Full Text] [PDF]
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M. Ridderstrale, E. Carlsson, M. Klannemark, A. Cederberg, C. Kosters, H. Tornqvist, H. Storgaard, A. Vaag, S. Enerback, and L. Groop FOXC2 mRNA Expression and a 5' Untranslated Region Polymorphism of the Gene Are Associated With Insulin Resistance Diabetes, December 1, 2002; 51(12): 3554 - 3560. [Abstract] [Full Text] [PDF]
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M. M. Nellis, C. B. Doering, A. Kasinski, and D. J. Danner Insulin increases branched-chain alpha -ketoacid dehydrogenase kinase expression in Clone 9 rat cells Am J Physiol Endocrinol Metab, October 1, 2002; 283(4): E853 - E860. [Abstract] [Full Text] [PDF]
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M. K. Dahle, L. M. Gronning, A. Cederberg, H. K. Blomhoff, N. Miura, S. Enerback, K. A. Tasken, and K. Tasken Mechanisms of FOXC2- and FOXD1-mediated Regulation of the RIalpha Subunit of cAMP-dependent Protein Kinase Include Release of Transcriptional Repression and Activation by Protein Kinase Balpha and cAMP J. Biol. Chem., June 14, 2002; 277(25): 22902 - 22908. [Abstract] [Full Text] [PDF]
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