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Originally published In Press as doi:10.1074/jbc.M705869200 on July 30, 2007

J. Biol. Chem., Vol. 282, Issue 39, 28549-28556, September 28, 2007
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Tumor Necrosis Factor Receptor-1 Can Function through a G{alpha}q/11-beta-Arrestin-1 Signaling Complex*

Yuji Kawamata1, Takeshi Imamura1, Jennie L. Babendure, Juu-Chin Lu, Takeshi Yoshizaki, and Jerrold M. Olefsky2

From the Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093-0673

Received for publication, July 17, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor-{alpha} (TNF{alpha}) is a proinflammatory cytokine secreted from macrophages and adipocytes. It is well known that chronic TNF{alpha} exposure can lead to insulin resistance both in vitro and in vivo and that elevated blood levels of TNF{alpha} are observed in obese and/or diabetic individuals. TNF{alpha} has many acute biologic effects, mediated by a complex intracellular signaling pathway. In these studies we have identified new G-protein signaling components to this pathway in 3T3-L1 adipocytes. We found that beta-arrestin-1 is associated with TRAF2 (TNF receptor-associated factor 2), an adaptor protein of TNF receptors, and that TNF{alpha} acutely stimulates tyrosine phosphorylation of G{alpha}q/11 with an increase in G{alpha}q/11 activity. Small interfering RNA-mediated knockdown of beta-arrestin-1 inhibits TNF{alpha}-induced tyrosine phosphorylation of G{alpha}q/11 by interruption of Src kinase activation. TNF{alpha} stimulates lipolysis in 3T3-L1 adipocytes, and beta-arrestin-1 knockdown blocks the effects of TNF{alpha} to stimulate ERK activation and glycerol release. TNF{alpha} also led to activation of JNK with increased expression of the proinflammatory gene, monocyte chemoattractant protein-1 and matrix metalloproteinase 3, and beta-arrestin-1 knockdown inhibited both of these effects. Taken together these results reveal novel elements of TNF{alpha} action; 1) the trimeric G-protein component G{alpha}q/11 and the adapter protein beta-arrestin-1 can function as signaling molecules in the TNF{alpha} action cascade; 2) beta-arrestin-1 can couple TNF{alpha} stimulation to ERK activation and lipolysis; 3) beta-arrestin-1 and G{alpha}q/11 can mediate TNF{alpha}-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic low grade inflammation is a critical feature of a number of common, non-immune diseases such as insulin resistance/obesity, type 2 diabetes mellitus, certain forms of cancer, and neurodegenerative disorders. The cytokine, TNF{alpha}3, is a key signaling molecule that activates proinflammatory effects in target cells. TNF{alpha} binds to the TNF receptor-1 (TNF-R1) and receptor-2 (TNF-R2) and stimulates the NF{kappa}B pathway through complexes that includes the TRAF, RIP (receptor-interacting protein), and TRADD (TNF-R-associated death domain) families (1, 2). Numerous studies have reported TNF{alpha} signaling to NF{kappa}B activation; however, the mechanisms underlying TNF{alpha} stimulates ERK, cdc42, or PI 3-kinase actually remain incompletely understood. It is well known that chronic TNF{alpha} treatment can lead to insulin resistance both in vitro (3) and in vivo (4), and elevated levels of TNF{alpha} have been reported in obese and/or diabetic individuals. Thus, further insights into the mechanisms of TNF{alpha} signaling are of interest. Recent findings reported that TNF-R1 is observed in plasma membrane microdomains, termed lipid rafts and/or caveolae (5, 6), in which various G-proteins, 7-transmembrane (TM) receptors (7-TMRs), and many other signaling components are collected. For example, the endothelin-1 (ET-1) receptor ETAR, a Gq-coupled 7-TM receptor, is detected in caveolae, and insulin receptors are also found in this membrane structure (7). Interestingly, it has been shown that the insulin receptor can couple into the heterotrimeric G-protein G{alpha}q/11 (8) and that chronic ET-1 treatment can induce insulin resistance by heterologous desensitization of G{alpha}q/11 signaling (9, 10).

Based on these facts, we sought to determine whether TNF{alpha} signaling could couple with G-protein components to mediate biologic effects. In this report we show that in 3T3-L1 adipocytes, TNF{alpha} signals through the TNF-R1·TRAF2 receptor complex and that this can couple into a trimeric G-protein G{alpha}q/11 and an adaptor protein beta-arrestin-1 to mediate TNF{alpha} activation of ERK, lipolysis, and stimulation of the proinflammatory pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Mouse monoclonal anti-phosphotyrosine (PY-20) antibody was purchased from BD Biosciences. Horseradish peroxidase-linked anti-rabbit, -mouse, -goat antibodies, anti-c-Jun N-terminal kinase (JNK), -cdc42, TRAF2, -p110{alpha} subunit of PI 3-kinase, and -pan-Src polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-specific anti-ERK and -JNK antibodies were from Cell Signaling, Inc. TNF-R1 antibody and mouse- and human-TNF{alpha} were from R&D Systems (Minneapolis, MN). Mouse TNF{alpha} was used in the experiments unless otherwise specified. Anti-G{alpha}q/11 antibody, Src-kinase inhibitor (PP2), PD98059, LY294002, and toxin-B were from Calbiochem. [{gamma}-32P]GTP{gamma}-azidoanilide was from Affinity Labeling Technologies, Inc. (Lexington, KY). RT-PCR primers were from Invitrogen, and the oligonucleotide sequences used were: matrix metalloproteinase 3 (MMP3) forward, 5'-TGACCCCACTCACTTTCTCC-3'; MMP3 reverse, 5'-GCCTTGGCTGAGTGGTAGAG-3'; monocyte chemoattractant protein-1 (MCP-1) forward, 5'-AGCACCAGCCAACTCTCAC-3'; MCP-1 reverse, 5'-TCTGGACCCATTCCTTCTTG-3'; TNF-R1 forward, 5'-GACCGGGAGAAGAGGGATAG-3'; TNF-R1 reverse, 5'-CACGCACTGGAAGTGTGTCT-3'; TNF-R2 forward, 5'-AAATGCAAGCACAGATGCAG-3'; TNF-R2 reverse, 5'-TCCTGGGATTTCTCATCAGG-3'. Cell culture materials and other radioisotopes were from ICN (Costa Mesa, CA). All other reagents were purchased from Sigma.

siRNA Transfection—3T3-L1 adipocytes (day 8 after differentiation) were transiently transfected by electroporation (GENE PULSER, Bio-Rad) with 2 nmol of siRNA duplex, as previously described (11). 24–120 h after electroporation, the efficiency of each siRNA was analyzed by RT-PCR or Western blotting every 24 h, and the time point for the maximum efficiency of each siRNA was determined. All experiments with siRNA were performed 48–96 h after electroporation or microinjection. SiGENOME SMARTpool siRNA against TRAF2 and all other custom siRNAs were purchased from Dharmacon Research Inc. (Lafayette, CO). The target sequences of the siRNAs were follows: beta-arrestin 1-A, 5'-GGCCTGTGGTGTGGATTAT-3'; beta-arrestin 1-B, 5'-AGCCTTCTGTGCTGAGAAC-3'; G{alpha}q-A, 5'-GCTGGTGTATCAGAACATC-3';G{alpha}q-B, 5'-TCCATATGTAGATGCAATA-3';G{alpha}11-A, 5'-ACTCACACTTGGTCGATTA-3';G{alpha}11-B, 5'-GTTGGTGTACCAGAACATC-3'; cdc42, 5'-GTTATCCACAGACAGATGT-3'; TNF-R1, 5'-GTATGTCCATTCTAAGAAC-3'; TNF-R2, 5'-ACTCCAAGCATCCTTACAT-3'. Each one of the paired siRNAs (A and B) was used separately, and this confirmed the absence of off-target effects. All sequences were confirmed to have no homology to any other genes by BLAST search (NCBI, National Institutes of Health).

Photoaffinity GTP-loading Assay—Photoaffinity GTP-labeling of membrane-associated G-proteins was performed as we described previously (12) with some modifications. Membrane fractions were semi-purified from scraped 3T3-L1 adipocytes (1 x 108 cells) by Dounce homogenization and centrifugation (18,000 x g, 10 min) in a buffer containing 50 mM HEPES (pH 7.4), 1 mM EDTA, 1 mM Na3VO4, 15 mM NaF, 1 mM phenylmethylsulfonyl fluoride and then immediately incubated with 10 µM [{gamma}-32P]GTP-azidoanilide (12.2 mCi/µmol) for 3 min followed by UV irradiation (254 nm) for 1 min on ice. Samples were lysed with the addition of Nonidet P-40 (final concentrations, 1%), then immunoprecipitated with anti-G{alpha}s, G{alpha}q/11, or G{alpha}i antibody. Immunoprecipitates were resolved by SDS-PAGE, and signals were quantitated by PhosphorImager (GE Healthcare).

Inositol Phosphate Production Assay—3T3-L1 adipocytes were incubated with myo-[3H]inositol (ICN, Costa Mesa, CA) (2 µCi/ml) in inositol-free Dulbecco's modified Eagle's medium for 16 h before assay. After washing cells twice with serum-free Dulbecco's modified Eagle's medium, cells were stimulated with TNF{alpha} (20 ng/ml) for 15 min. The accumulation of total 3H-labeled (IP1 and IP2 plus IP3) was measured as previously described (13).

cdc42 Assay—cdc42 activity was measured according to the manufacturer's instructions (Upstate%20Biotechnology">Upstate Biotechnology, Inc., Lake Placid, NY). 3T3-L1 adipocytes were starved for 16 h and stimulated with 17 nM insulin or 20 ng/ml TNF{alpha} for the indicated time periods, washed once with ice-cold phosphate-buffered saline, and lysed with lysis buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% IGEPAL CA-630, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 25 mM NaF for 15 min at 4 °C. Insoluble materials were removed by centrifugation. For a negative control cell lysate was incubated with 1 mM GDP for 15 min at 30 °C. 5 µg of p21-activated kinase 1-agarose beads, which specifically bound to active cdc42, were added to the cell lysates and incubated for 1 h at 4 °C. Agarose beads were washed with lysis buffer three times and boiled in Laemmli sample buffer. Samples were resolved by SDS-PAGE and immunoblotted with anti-cdc42 antibody.

Lipolysis Assay—Triglyceride hydrolysis (lipolysis) was measured using free glycerol reagent (Sigma) according to the manufacturer's specifications. Briefly, 3T3-L1 adipocytes were washed twice and incubated with L-buffer (Dulbecco's modified Eagle's medium without phenol red plus 0.1% bovine serum albumin) then stimulated with or without TNF{alpha} (20 ng/ml) for 16 h. To measure lipolysis, 150 µl of free glycerol assay reagent was incubated with 10 µl of culture supernatants and glycerol standards for 15 min, and the absorbance was read at 540 nm. A standard curve constructed from the glycerol standards was used to calculate the concentration of glycerol in the culture supernatants.

Cell Culture and Other Methods—3T3-L1 adipocyte cell culture (8), immunoprecipitation with dithiobis(succinimidyl propionate) cross-linking (12), Src kinase assay (14), RT-PCR and PI 3-kinase assay (15), arachidonic acid release assay (16), and cyclic AMP assay (17) were performed as we previously described.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The G-protein Signaling Component, beta-Arrestin-1, Couples with the TNF Receptor Complex—beta-Arrestin-1 is a scaffold protein that functions in a number of 7-TMR (typical G protein-coupled receptor) signaling pathways, and TRAF2 serves as an adaptor protein for TNF{alpha} signaling. To assess the relationship between these systems, we examined the association between beta-arrestin-1 and TRAF2 in FLAG-tagged beta-arrestin-1 expressing 3T3-L1 adipocytes. As shown in Fig. 1A, beta-arrestin-1 was detected in anti-TRAF2 antibody immunoprecipitates in the basal state, and this was greatly reduced after TNF{alpha} stimulation (*, p < 0.05; **, p < 0.01). This result indicates that beta-arrestin-1 associates with TNF receptor complexes in the basal state and dissociates from this complex after TNF{alpha} stimulation. In the 7-TMR systems, beta-arrestin-1 binds to Src kinase, and ligand stimulation results in Src-kinase activation (18). To see if this was also the case for TNF{alpha}, we measured Src-kinase activity, and we detect that, like ET-1, TNF{alpha} acutely stimulated Src-kinase activity (2.5-fold above basal), as shown in Fig. 1B. Using RNA interference, electroporation with siRNA against beta-arrestin-1 (betaar1-A or betaar1-B) decreased protein expression in 3T3-L1 adipocytes (Fig. 1C), and we found that acute stimulation of Src kinase activity with TNF{alpha} was inhibited by siRNA knockdown of beta-arrestin-1 (Fig. 1D). These results suggest that TNF{alpha}-induced Src-kinase activation is dependent on beta-arrestin-1, similar to 7-TMR signaling.


Figure 1
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  		FIGURE 1.
TNF{alpha} stimulates Src-kinase activity to phosphorylate G{alpha}q/11 via beta-arrestin-1 in 3T3-L1 adipocytes. A, serum-starved 3T3-L1 adipocytes expressing either FLAG-tagged beta-arrestin-1 (FL) or enhance green fluorescent protein control (Ctr.) were stimulated with TNF{alpha} (20 ng/ml) for the indicated time periods. Cells were then washed twice with ice-cold phosphate-buffered saline and incubated with dithiobis(succinimidyl propionate) cross-linker for 1 h on ice, as described under "Experimental Procedures." The resulting cell lysates were immunoprecipitated with control IgG or anti-TRAF2 antibody (Ab), and immunoprecipitation (IP) samples were analyzed by Western blotting with biotin-conjugated anti-FLAG antibody. The IP efficiency of TRAF2 antibody was the same in each lane. The result shown in the lower panel is a representative image from three independent experiments, and the scanned bar graph (upper panel) shows % maximum of the basal condition (mean ± S.E. of three independent experiments). WCL, whole cell lysates from cells expressing FLAG-tagged beta-arrestin-1. Asterisk, statistical significance (*, p < 0.05; **, p < 0.01) in a two-tailed Student t test compared with basal conditions. B, serum-starved 3T3-L1 adipocytes were stimulated with insulin (Ins, 100 ng/ml), ET-1 (10 nM), or TNF{alpha} (2, 20, or 100 ng/ml) for 3 min. Cell lysates were immunoprecipitated with anti-pan Src antibody, and Src-kinase activity in the IP samples was assayed for the ability to phosphorylate the Src -kinase substrate peptide, as described under "Experimental Procedures." Data are presented as the mean ± S.E. of three independent experiments. C, differentiated 3T3-L1 adipocytes were electroporated with specific siRNA against beta-arrestin-1 (betaar1-A or betaar1-B) or a scrambled sequence (Scr). 24–120 h after electroporation, protein expression of beta-arrestin-1 were analyzed by Western blotting every 24 h, and the maximum efficiency of siRNA was observed during 24–96 h after the transfection. The images shown are representative results from three independent experiments. The scanned bar graphs are shown as % maximum of control (Scr) siRNA-transduced cells (mean ± S.E. of three independent experiments). D, 3T3-L1 adipocytes transduced with siRNA against beta-arrestin-1 (betaar1) were serum-starved for 4 h then stimulated with TNF{alpha} for 3 min. Src kinase activity was measured as described above. Data are represented as % maximum of scrambled (Scr) siRNA-transfected cells (mean ± S.E. of three independent experiments). E, 3T3-L1 adipocytes were incubated for 96 h after transduction with siRNA against beta-arrestin-1 (betaar1-A or betaar1-B) or scrambled sequence (Scr) as a control, then cells were serum-starved for 4 h and stimulated with or without TNF{alpha} (20 ng/ml) for 3 min. Cell lysates were immunoprecipitated with control IgG or anti-phosphotyrosine (PY20) antibody, and immunoprecipitates were analyzed by Western blotting with anti-G{alpha}q/11 antibody. The result shown in the lower panel is a representative image from three independent experiments, and the scanned bar graph (upper panel) shows % maximum of control (Scr) siRNA-transduced cells with TNF{alpha} stimulation (mean ± S.E. of three independent experiments). Asterisk, statistical significance (p < 0.01) versus control in a two-tailed Student t test. F, serum-starved 3T3-L1 adipocytes were pre-incubated with the Src kinase inhibitor, PP2 (400 nM), or Me2SO (DMSO) vehicle for 30 min, then stimulated with TNF{alpha} (20 ng/ml, 3 min) or insulin (100 ng/ml, 1 min). Tyrosine phosphorylation of G{alpha}q/11 was analyzed as described above. The scanned bar graphs (top panel) are shown as % maximum of TNF{alpha}-stimulated cells pretreated with Me2SO (mean ± S.E. of three independent experiments). Asterisk, statistical significance (p < 0.01) in a two-tailed Student t test comparing PP2 and control (Me2SO) treatment with TNF{alpha} stimulation.

 
Umemori et al. (19) reported that the activation of G{alpha}q/11 is coincident with phosphorylation of tyrosine residue 356 in the C terminus of G{alpha}q/11. In response to ET-1 stimulation, we have previously reported that Src kinase plays a key role, leading to tyrosine phosphorylation of G{alpha}q/11 (14). Accordingly, we examined the effects of TNF{alpha} on G{alpha}q/11 and found that TNF{alpha} acutely induced tyrosine phosphorylation of G{alpha}q/11 at 3 min after stimulation and that this effect was inhibited by siRNA-mediated beta-arrestin-1 knockdown (Fig. 1E). Because two different siRNA against beta-arrestin-1 (betaar1-A or betaar1-B in the upper panel) showed quite comparable results, this confirms that off-target effects do not contribute to our results. Furthermore, the Src-kinase inhibitor, PP2, also inhibited TNF{alpha}-induced G{alpha}q/11 tyrosine phosphorylation (Fig. 1F), similar to its effects to block ET-1-stimulated phosphorylation (14). Interestingly, PP2 did not inhibit insulin-induced G{alpha}q/11 phosphorylation, suggesting that the insulin receptor kinase itself can phosphorylate G{alpha}q/11 independent of Src kinase activity. These results indicate that beta-arrestin-1 mediates the effect of TNF{alpha} to phosphorylate G{alpha}q/11 via the activation of Src-kinase.

TNF{alpha} Stimulates G{alpha}q/11 Activity without Leading to Phospholipase Cbeta (PLC-beta) Activation—To confirm the activation of G{alpha}q/11, we performed a GTP-loading assay using photoaffinity GTP labeling method, as described under "Experimental Procedures." As shown in Fig. 2A, TNF{alpha} stimulation increased the amount of GTP bound to G{alpha}q/11 (3-fold) but had little effect on GTP loading of G{alpha}i or G{alpha}s. Consistent with this, TNF{alpha} stimulation did not lead to changes in the cellular cyclic AMP level (Fig. 2B). It is generally thought that activated G{alpha}q/11 stimulates PLC-beta, leading to the induction of diacylglycerol, inositol phosphate-3, and subsequent arachidonic acid release. We measured the total amount of inositol phosphates (Fig. 2C, left panel) and arachidonic acid release (Fig. 2C, right panel) in 3T3-L1 adipocytes and found that TNF{alpha} stimulation interestingly did not increase either of these metabolites, suggesting that TNF{alpha} does not activate PLC-beta. This is also observed in insulin receptor signaling (Fig. 2C), in which G{alpha}q/11 is activated (8), and importantly, both receptors do not have a 7-TM type structure. Several studies have revealed that the second and/or third intracellular loops of 7-TM receptors are required for PLC-beta activation (20, 21), and these motifs are not present in the insulin and TNF receptors. Therefore, we hypothesized that non-7-TM type receptor cannot associate with PLC-beta. We tested the co-immunoprecipitation with receptor antibody and found no co-immunoprecipitation of PLC-beta3 (the main isoform of the PLC-beta family in 3T3-L1 adipocytes) with anti-TNF-R1 antibody, although PLC-beta3 was modestly co-immunoprecipitated with anti-ETAR-Ab after ET-1 treatment (Fig. 2D).


Figure 2
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  		FIGURE 2.
TNF{alpha} stimulates G{alpha}q/11 activity without PLC-beta activation in 3T3-L1 adipocytes. A, after photoaffinity labeling with [{gamma}-32P]GTP-azidoanilide, solubilized membranes were immunoprecipitated with anti-G{alpha}s, G{alpha}q/11, or G{alpha}i antibody. Immunoprecipitates (IP) were analyzed by SDS-PAGE and PhosphorImager, as described under "Experimental Procedures." Data were quantitated by PhosphorImager and represent the mean ± S.E. of three or four independent experiments (upper panel). A representative image is shown in lower panel. B, serum-starved 3T3-L1 adipocytes were stimulated with either TNF{alpha} (20 ng/ml), isoproterenol (ISO, 10 µM), ET-1 (10 nM), or insulin (100 ng/ml) for 15 min. Intracellular cyclic AMP levels were normalized with cellular protein concentrations. Data are presented as -fold over basal (mean ± S.E. of three independent experiments). C, 3T3-L1 adipocytes were incubated with [3H]inositol (left panel) or [3H]arachidonic acid (right panel) for 16 h, then washed and stimulated with either TNF{alpha} (20 ng/ml), ET-1 (10 nM), or insulin (100 ng/ml) for 15 min. 3H levels in the samples were quantitated and normalized for cellular protein concentration, as described in under "Experimental Procedures." Data are presented as -fold basal (mean ± S.E. of three independent experiments). D, differentiated 3T3-L1 adipocytes were serum-starved for 4 h and stimulated with TNF{alpha} (20 ng/ml) or ET-1 (10 nM) for the indicated time periods. Cell lysates were immunoprecipitated with control (Ctrl.) IgG, anti-ETAR, or TNF-R1 antibody, and IP samples were analyzed by Western blotting with anti-PLC-beta3 antibody. The IP efficiency of anti-TNF-R1 or ETAR antibody was the same in each lane (data not shown). The representative image is from two independent experiments. WCL, whole cell lysates.

 
Biological Roles of beta-Arrestin-1-G{alpha}q/11 as Signaling Mediators for TNF{alpha} Action—To investigate which biological effects of TNF{alpha} are mediated by beta-arrestin-1/Src kinase and G{alpha}q/11, we first focused on JNK, a proinflammatory pathway mediator, since the mechanism of JNK activation is still unclear. Using RNA interference-induced knockdown of TNF-R1, TNF-R2, or TRAF2 (Fig. 3, A and B, left panel), we found that TNF{alpha} (20 ng/ml, 8 min) stimulated JNK phosphorylation and that this effect was strongly inhibited by TNF-R1 or TRAF2 knockdown but not by TNF-R2 knockdown (Fig. 3C), consistent with the view that a TNF-R1·TRAF2 complex mediates TNF{alpha} signaling to JNK activation. It has been reported previously that activation of JNK is mediated by cdc42 (22) and PI 3-kinase (23) and that TNF{alpha} can stimulate cdc42 (24) and PI 3-kinase activities (25), although the mechanisms for activation of these kinases are unclear. Because activated G{alpha}q/11 can lead to stimulation of cdc42 and cdc42-associated PI 3-kinase (26), we assessed this pathway with respect to TNF{alpha} stimulation. Using RNA interference showing in Figs. 1C and 3B, we found that TNF{alpha}-stimulated JNK phosphorylation was inhibited by beta-arrestin-1, G{alpha}q/11, or cdc42 knockdown in 3T3-L1 adipocytes (Fig. 3D). Two different siRNAs against beta-arrestin-1 (betaar1-A in Fig. 3D, left panels, and betaar1-B in rightside panels) or G{alpha}q/11 (Gq-A + G11-A in Fig. 3D, left panels, and Gq-B + G11-B in right side panels) showed quite similar results, indicating that there were no off-target effects in these siRNAs.

To confirm these signaling events, we tested the effect of kinase inhibitors on TNF{alpha} signaling. Consistent with the above results, TNF{alpha}-stimulated JNK activation was inhibited by pretreatment of the cells with the Src inhibitor (PP2), the PI 3-kinase inhibitor (LY294002, LY), or the cdc42 inhibitor (toxin-B, TB) but not by MEK inhibitor (PD98059, PD) (Fig. 4A). Furthermore, we confirmed that TNF{alpha} stimulates cdc42 activity (Fig. 4B) and cdc42-associated PI 3-kinase activity (Fig. 4C), and these effects were inhibited by PP2 treatment (*, p < 0.01) (Fig. 4, B and C). Taken together, these data indicated that TNF{alpha} signaling goes through the TNF-R1·TRAF2 -> beta-arrestin-1-Src-kinase -> G{alpha}q/11 -> cdc42/PI 3-kinase pathway to activate JNK, a proinflammatory pathway in 3T3-L1 adipocytes.

It has been reported that TNF{alpha}-induced ERK activation was regulated by Src kinase (27), although the full mechanisms of ERK activation remain unclear. In 3T3-L1 adipocytes TNF{alpha}-induced ERK phosphorylation was maximal at 15 min after stimulation, and this effect was significantly inhibited by TNF-R1 or TRAF2 knockdown but not the results for TNF-R2 knockdown (Fig. 5A), similar to the results for JNK activation (Fig. 3B). Previous reports showed that human TNF{alpha} can bind to mouse TNF-R1 but not to mouse TNF-R2 (28), and this is fully consistent with the results in Fig. 5A, showing that the effects of human TNF{alpha} on ERK phosphorylation are also blocked by siRNA-mediated TNF-R1 knockdown. As seen in Fig. 5B, TNF{alpha}-induced ERK phosphorylation was decreased 68% by beta-arrestin-1 knockdown (*, p < 0.01), indicating that beta-arrestin-1 mediates the effect of TNF-R1 signaling. Interestingly, neither G{alpha}q/11 knockdown nor inhibition of G{alpha}i with pertussis toxin treatment inhibited TNF{alpha}-stimulated ERK phosphorylation (Fig. 5B), demonstrating that this signaling pathway does not utilize these G-protein components. This is of particular interest since Figs. 1E and 2A showed that TNF{alpha} can lead to G{alpha}q/11 phosphorylation and activation, indicating a separation of signaling pathways which flow through beta-arrestin-1 versus beta-arrestin-1-G{alpha}q/11.


Figure 3
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  		FIGURE 3.
TNF-R1 signaling engages beta-arrestin-1-G{alpha}q/11 to activate JNK. A, differentiated 3T3-L1 adipocytes were electroporated with specific siRNA against TNF-R1, TNF-R2, or a scrambled control (Scr). 24–120 h after electroporation, mRNA of the target genes were analyzed by RT-PCR every 24 h, and the time point for the maximum efficiency of each siRNA was determined. The images shown are representative results from three independent experiments. The scanned bar graphs are shown as % maximum of control (Scr) siRNA-transduced cells (mean ± S.E. of three independent experiments). B, differentiated 3T3-L1 adipocytes were electroporated with specific siRNAs against TRAF2 (left panels), G{alpha}q + G{alpha}11 (middle panels), cdc42 (right side panels), or a scrambled control (Scr). The maximum efficiency of each siRNA was determined as described in A. The images shown are representative results from three independent experiments. The scanned bar graphs are shown as % maximum of control (Scr) siRNA-transduced cells (mean ± S.E. of three independent experiments). C, 48 h after transduction with specific siRNA against TNF-R1, TNF-R2, TRAF2, or scrambled (Scr) control, serum-starved 3T3-L1 adipocytes (day 10 after differentiation) were stimulated with TNF{alpha} (20 ng/ml) for 8 min, and cell lysates were analyzed by Western blotting with anti-JNK (bottom panel) or phospho (P)-specific JNK antibody (middle panel). The scanned bar graphs (top panel) are shown as % maximum of scrambled (Scr) siRNA-transduced cells (mean ± S.E. of three independent experiments). Asterisk, statistical significance (p < 0.01) versus control in a two-tailed Student t test. D, serum-starved 3T3-L1 adipocytes (day 10) transduced with specific siRNA against beta-arrestin-1 (betaar1-A in left panels, and betaar1-B in right panels), G{alpha}q/11 (Gq-A + Gq-A in left panels and Gq-B + G11-B in right panels), cdc42, or scrambled (Scr) control were analyzed as above. The scanned bar graphs (top panel) are shown as % maximum of scrambled (Scr) siRNA-transduced cells (mean ± S.E. of three independent experiments). Asterisk, statistical significance (p < 0.05) versus control in a two-tailed Student t test.

 
In other systems we (29) and others (30) have shown that beta-arrestin-1 is an upstream regulator of ERK activity, and recent reports by Kang et al. (31) revealed that beta-arrestin-1 can be translocated to the nucleus after ligand stimulation, where it participates in regulation of gene expression. Accordingly, we measured mRNA expression levels of two TNF{alpha} target genes, MMP3 and MCP-1, and found that expression of these genes was strongly stimulated by TNF{alpha} treatment (Fig. 5C). Because TNF{alpha} stimulation of both ERK and JNK1 appears to be beta-arrestin-1-dependent and because both ERK and JNK1 can regulate gene expression, we investigated the effects of beta-arrestin-1 knockdown and found that TNF{alpha}-induced MCP-1 and MMP3 expression was inhibited by depletion of beta-arrestin-1 (*, p < 0.03).

Another biologic effect of TNF{alpha} in adipocytes is stimulation of lipolysis. We found a role for beta-arrestin-1 signaling in this process by showing that TNF{alpha}-induced lipolysis was partially inhibited by beta-arrestin-1 knockdown (*, p < 0.05) but not by G{alpha}q/11 knockdown, as seen in Fig. 5D, left panel. Because beta-arrestin-1 mediates TNF{alpha}-induced ERK activation, we treated the cells with the MEK inhibitor PD98059 and found that this also partially inhibited TNF{alpha}-induced lipolysis (*, p < 0.05) (Fig. 5D, right panel), consistent with a previous report (32).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
TNF{alpha} stimulation induces a wide range of biologic effects related to innate immunity, inflammation, insulin resistance/diabetes, and other conditions. Chronic inflammation is increasingly recognized as a major cause of decreased insulin sensitivity. By activating pro-inflammatory pathways (33), TNF{alpha} can cause insulin resistance in vitro (3) and in animals treated with TNF{alpha} (4). The molecular mechanisms of TNF{alpha} action have been extensively studied, and many elements of this pathway have been defined (2). In this study we have identified new elements of a novel TNF{alpha} signaling pathway in adipocytes, which flows through a TNF-R1·TRAF2 complex and includes the use of the classical 7-TMR signaling components, beta-arrestin-1, and G{alpha}q/11. We found that beta-arrestin-1 can mediate TNF{alpha} effects on Src kinase and ERK activation and that G{alpha}q/11 can couple TNF{alpha} action to cdc42, PI 3-kinase, and JNK activation, leading to the induction of lipolysis and proinflammatory gene expression in 3T3-L1 adipocytes.


Figure 4
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  		FIGURE 4.
TNF-R1 signaling activates JNK through cdc42/PI 3-kinase pathway. A, serum-starved 3T3-L1 adipocytes were pretreated with LY294002 (LY, 10 µM, 30 min), toxin-B (TB, 50 ng/ml, 2 h), PP2 (400 nM, 30 min), PD98059 (PD, 25 µM, 30 min), or Me2SO (DMSO) vehicle for 30 min, then stimulated with or without TNF{alpha} (20 ng/ml, 8 min). Cell lysates were analyzed, and the results were shown as described in Fig. 3B. The scanned bar graphs (top panel) are shown as % maximum of TNF{alpha}-stimulated cells pretreated with Me2SO (mean ± S.E. of three independent experiments). Asterisk, statistical significance (p < 0.01) in a two-tailed Student t test comparing with control (Me2SO) treatment. B, cdc42 activity was measured by the association with GST-p21-activated kinase 1 beads, which specifically recognize activated cdc42. Serum-starved 3T3-L1 adipocytes were pretreated with or without PP2 (400 nM, 30 min) and stimulated with TNF{alpha} (20 ng/ml) for 3 min. Cell lysates were incubated with GST-p21-activated kinase 1 beads, and the precipitates were analyzed by Western blotting with anti-cdc42 antibody (bottom panel). The scanned bar graphs represent the mean ± S.E. of three independent experiments. C, 3T3-L1 adipocytes were serum-starved for 6 h, then pretreated with 400 nM PP2 or 0.1% Me2SO vehicle for 30 min before stimulation with TNF{alpha} (20 ng/ml) for 5 min. PI 3-kinase activity in the immunoprecipitates (insets) with anti-cdc42 (left panel) or -p110{alpha} antibody (right panel) was measured as described under "Experimental Procedures." Data were quantitated by PhosphorImager and represent the mean ± S.E. of three or four independent experiments. Asterisk, statistical significance (p < 0.01) versus control in a two-tailed Student t test.

 
It is well known that beta-arrestin-1 plays several roles in 7-TMR function, including receptor desensitization and internalization (34). Interestingly, it has also been reported that beta-arrestin-1 interacts with non-7-TM type receptors, such as the insulin-like growth factor-I, epidermal growth factor, and insulin receptors (35), and here we show that the TNF-R1·TRAF2 complex can signal through beta-arrestin-1 to mediate TNF{alpha} biological effects in 3T3-L1 adipocytes. As such, these studies further demonstrate the diverse functions of beta-arrestin-1 as an adaptor protein mediating the signaling properties of receptor systems distinct from its classical role in 7-TMR action. Our studies also show that the G-protein component, G{alpha}q/11, can also function in the TNF{alpha} signaling pathway. To accomplish this task, TNF{alpha} signaling uses beta-arrestin-1 effects to activate Src kinase, causing G{alpha}q/11 tyrosine phosphorylation and activation. Together these effects can mediate downstream signaling events such as activation of ERK, PI 3-kinase, and lipolysis.

We investigated the downstream TNF{alpha} effects transduced by beta-arrestin-1 and/or G{alpha}q/11, and one of them is JNK activation. It is well known that JNK can propagate the effects of TNF{alpha} to induce proinflammatory gene expression, and this can result in insulin resistance. Previous studies have shown that TNF{alpha}-induced JNK activation is mediated by TRAF2 (36), cdc42 (22), Src (37), and PI 3-kinase (38). There may be multiple ways in which TNF{alpha} can cause JNK activation, and consistent with these reports, our findings show that TNF{alpha} can signal through beta-arrestin-1/Src-kinase -> G{alpha}q/11 -> cdc42/PI 3-kinase to activate JNK. Similarly, previous studies revealed multiple mediators for TNF{alpha}-induced ERK activation, including TRAF2, MADD, and Src kinase (27, 39). Ligand stimulation can induce diverse signaling pathways leading to ERK activation; e.g. insulin-like growth factor-I receptor can signal through beta-arrestin-1/G{alpha}i as well as SHC to activate ERK (30, 40). Consistent with this formulation, siRNA-induced beta-arrestin-1 knockdown did not completely inhibit TNF{alpha}-induced ERK phosphorylation (Fig. 5B), indicating the presence of another pathway(s) from the TNF receptor to this biologic end point. Because NF{kappa}B pathway may also be involved in lipolytic effects (41), this may also explain why beta-arrestin-1 knockdown only partially inhibits TNF{alpha}-stimulated lipolysis (Fig. 5D).

Previous studies have shown that beta-arrestin-1 is an upstream mediator of ERK activation in various receptor signaling systems (42) and that beta-arrestin-1 can translocate into the nucleus to exert transcriptional effects (31). In addition, activated JNK stimulates c-Jun, which translocates into the nucleus as a transcription factor activating pro-inflammatory gene expression (43). These findings suggested that beta-arrestin-1 might play a role in TNF{alpha}-induced gene expression. Consistent with this, our results show that siRNA-knockdown of beta-arrestin-1 exerts inhibitory effects on TNF{alpha}-stimulated MCP-1 and MMP3 gene expression.


Figure 5
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  		FIGURE 5.
Effects of TNF{alpha} on ERK, lipolysis, and gene expression are beta-arrestin-1 dependent. A, 48 h after transduction with specific siRNA against TNF-R1 (R1), TNF-R2 (R2), TRAF2, or scrambled (Scr) sequence, serum-starved 3T3-L1 adipocytes (day 10) were stimulated with mouse TNF{alpha} (20 ng/ml), human TNF{alpha} (20 ng/ml), or ET-1 (10 nM) for 15 min, and cell lysates were analyzed by Western blotting with anti-ERK (middle panel) or phospho (P)-specific ERK antibody (top panel). The scanned bar graphs (bottom panel) are shown as % maximum of scrambled (Scr) siRNA-transduced cells (mean ± S.E. of three independent experiments). B, 3T3-L1 cells transduced with specific siRNAs against beta-arrestin-1 (betaar1-A), G{alpha}q/11 (Gq-A + G11-A) or a scrambled (Scr) sequence (left panel), or pretreated with pertussis toxin (PTX, 100 ng/ml, 4 h) (right panel) were stimulated with mouse TNF{alpha} (20 ng/ml, 15 min). Cell lysates were analyzed, and the results were shown as described above. C, total RNA samples were purified from serum-starved 3T3-L1 adipocytes stimulated with or without mouse TNF{alpha} (20 ng/ml) for 2 or 4 h after transduction with siRNA against beta-arrestin-1 (betaar1-A) or scrambled (Scr) sequence. The expression levels of MMP3, MCP-1, and beta-actin were analyzed by RT-PCR. Data were adjusted to the expression level of beta-actin as an internal control and are shown as % maximum control (mean ± S.E. of four independent experiments). D, 3T3-L1 adipocytes transduced with siRNA as shown above (B) or pretreated with PD98059 (PD, 25 µM, 30 min) or Me2SO (DMSO) vehicle were stimulated with mouse TNF{alpha} (20 ng/ml) for 15 h. Glycerol concentration was measured in the resulting culture supernatant, normalized to cellular protein concentration. Data represented the mean ± S.E. of three or five independent experiments. Asterisk, statistical significance versus control in a two-tailed Student t test.

 
TNF{alpha} stimulation induces a wide range of biologic effects related to innate immunity, inflammation, insulin resistance/diabetes, and other conditions. Chronic inflammation is increasingly recognized as a major cause of decreased insulin sensitivity, and by activating proinflammatory pathways (33), TNF{alpha} can cause insulin resistance in vitro (3) and in animals treated with TNF{alpha} (4). Last, it is well known that elevated circulating free fatty acid levels can be a cause of insulin resistance (44). In this study we have identified new elements in TNF{alpha} signaling pathways in adipocytes, which can lead to inflammatory pathway activation and lipolysis (Fig. 6). This pathway flows through a TNF-R1·TRAF2 receptor complex and includes the use of classical 7-TMR signaling components, beta-arrestin-1 and G{alpha}q/11, showing a novel signaling paradigm for these molecules distinct from their traditional functions mediating signals from an array of 7-TMRs.


Figure 6
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  		FIGURE 6.
Schematic model of TNF{alpha} signaling via beta-arrestin 1-G{alpha}q/11-cdc42 pathway. TRADD, TNFR-associated death domain; RIP, serine and threonine protein kinase receptor-interacting protein; TRAF2, TNFR-associated factor 2; NF{kappa}B, nuclear factor-{kappa}B; IKK, inhibitor of I{kappa}B kinase; p38, p38 mitogen-activated protein kinase.

 

   FOOTNOTES
 
* This work was supported by National Institutes of Health Research Grant DK 33651, University of California Discovery Program Project bio03-10383 (BioStar), and United States and Israel Binational Scientific Foundation Grant 2003238. 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. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: University of California, San Diego, Dept. of Medicine (0673), 9500 Gilman Dr., La Jolla, CA 92093. Tel.: 858-534-6651; Fax: 858-534-6653; E-mail: jolefsky{at}ucsd.edu.

3 The abbreviations used are: TNF, tumor necrosis factor; TNF-R, TNF receptor; 7-TMR, 7-transmembrane (TM) receptor; ET-1, endothelin-1; TRAF2, TNF receptor-associated factor 2; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; MMP3, matrix metalloproteinase 3; MCP-1, monocyte chemoattractant protein-1; PI 3-kinase, phosphatidylinositol 3-kinase; IP, immunoprecipitation; betaar, beta-arrestin; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PLC, phospholipase C; RT, reverse transcription; siRNA, small interfering RNA. Back


   ACKNOWLEDGMENTS
 
We thank Elizabeth J. Hansen for editorial assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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