Selective Activation of Mitogen-Activated Protein (MAP) Kinase Kinase 3 and p38{alpha} MAP Kinase Is Essential for Cyclic AMP-Dependent UCP1 Expression in Adipocytes

The sympathetic nervous system regulates the activity and expressionof uncoupling protein 1 (UCP1) through the three ß-adrenergicreceptor subtypes and their ability to raise intracellular cyclicAMP (cAMP) levels. Unexpectedly, we recently discovered thatthe cAMP-dependent regulation of multiple genes in brown adipocytes,including Ucp1, occurred through the p38 mitogen-activated proteinkinases (MAPK) (W. Cao, K. W. Daniel, J. Robidoux, P. Puigserver,A. V. Medvedev, X. Bai, L. M. Floering, B. M. Spiegelman, andS. Collins, Mol. Cell. Biol. 24:3057-3067, 2004). However, nowell-defined pathway linking cAMP accumulation or cAMP-dependentprotein kinase (PKA) to p38 MAPK has been described. Therefore,in the present study using both in vivo and in vitro models,we have initiated a retrograde approach to define the requiredcomponents, beginning with the p38 MAPK isoforms themselvesand the MAP kinase kinase(s) that regulates them. Our strategyincluded ectopic expression of wild-type and mutant kinasesas well as targeted inhibition of gene expression using smallinterfering RNA. The results indicate that the ß-adrenergicreceptors and PKA lead to a highly selective activation of thep38 ${alpha}$ isoform of MAPK, which in turn promotes Ucp1 gene transcription.In addition, this specific activation of p38 ${alpha}$ relies solely onthe presence of MAP kinase kinase 3, despite the expressionin brown fat of MKK3, -4, and -6. Finally, of the three scaffoldproteins of the JIP family expressed in brown adipocytes, onlyJIP2 coimmunoprecipitates p38 ${alpha}$ MAPK and MKK3. Therefore, in thebrown adipocyte the recently described scaffold protein JIP2assembles the required factors MKK3 and p38 ${alpha}$ MAPK linking PKAto the control of thermogenic gene expression.

INTRODUCTION

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Introduction

Uncoupling protein 1 (UCP1) is essential for rodents and othersmall mammals to maintain their body temperatures, since itis the sole mediator of cold-induced nonshivering thermogenesis(4, 6, 48); UCP1 is also a key contributor to the regulationof diet-induced thermogenesis (6, 58). The UCP1 protein resideswithin the inner membrane of mitochondria, where it serves asa portal for dissipation of the proton gradient such that respirationis uncoupled from ATP production and generates heat (35, 49,54). The UCP1 mRNA and protein are found in "brown" and to alesser extent in "white" adipose tissue; however, its expressionis confined to brown adipocytes (53). Similar brown adipocytesexist scattered within white adipose depots in adult humans(22, 37), but their contribution to thermogenesis is admittedlymodest. Nevertheless, studies in animals or humans exposed tohigh catecholamine levels or treated with sympathomimetics showthat brown adipocytes expressing UCP1 can be recruited withinwhite adipose depots (10, 12, 13, 16, 29).

Brown adipose tissue (BAT) and white adipose tissue are innervatedby sympathetic noradrenergic nerves (2, 3, 42, 50, 63). In responseto cold exposure or diet, sympathetic nervous system activationleads to the release of norepinephrine to interact with adrenergicreceptors (AR); in particular the family of ßARs (39,49, 55, 72). Catecholamine stimulation of the three ßARspresent in adipocytes promotes a series of events initiatedby the production of cyclic AMP (cAMP) and the activation ofcAMP-dependent protein kinase (PKA) (20, 56, 64). These eventsresult in lipolysis and liberation of free fatty acids (FFA)from triglyceride stores (39). These FFA serve not only as substratesfor oxidative respiration but also as allosteric activatorsof UCP1 function (24, 25, 60). ßAR-mediated increasesin cAMP also stimulate Ucp1 gene transcription. The cAMP responseof the Ucp1 gene is achieved predominantly through an enhancerregion (9, 15, 38). This enhancer, which is well conserved amongspecies (11), confers specificity of expression to brown adipocytesas well as the cAMP response and contains at least two key elements:a peroxisome proliferator response element (PPRE) and a cAMPresponse element (CRE).

We have recently shown that the cAMP-dependent transcriptionof the Ucp1 gene is regulated through these two elements byp38 mitogen-activated protein kinase (MAPK) (7). The effectof p38 MAPK on these elements occurs in a coordinated fashion.First, p38 MAPK phosphorylates a protein called PGC-1 ${alpha}$ (7), whichis a transcriptional coactivator and mediator of mitochondriogenesis(68), among other functions. This modification of PGC-1 ${alpha}$ enhancesits activity as a nuclear coactivator of gene transcriptionin coordination with peroxisome proliferator-activated receptor ${gamma}$ (PPAR ${gamma}$ ); PPAR ${gamma}$ in turn binds to the UCP1 PPRE (7). Second, p38directly stimulates expression of the Ucp1 gene through phosphorylationof the transcription factor ATF-2; ATF-2 binds to the CRE2 (7).Finally, the PGC-1 ${alpha}$ gene itself also possesses a CRE (28) butin the brown adipocyte is a target of p38-activated ATF-2 andnot CREB (7). By increasing the overall amount of PGC-1 ${alpha}$ proteinover time, p38 MAPK primes the cell for a sustained enhancementof UCP1 expression. Despite this new understanding of the roleof p38 MAPK in the regulation of the Ucp1 and PGC-1 ${alpha}$ genes inbrown fat, the cascade of signaling events downstream of PKAby which p38 MAPK becomes activated is completely unknown.

To begin to unravel this new pathway, we realized that it wasnecessary to tackle this problem in a "bottom-up" approach.Therefore, we reasoned that a strategy that would best servethis effort should first identify the actual p38 MAPK isoform(s)involved and proceed in a retrograde manner. The p38 MAPK groupis composed of four isoforms: p38 ${alpha}$ (26, 41), p38ß (32),p38 ${gamma}$ (43), and p38 ${delta}$ (66). Among them, p38 ${alpha}$ and -ß aresensitive to the pyrimidyl imidazoles SB202190 and SB205380(14, 23). These two isoforms are expressed in adipocytes (36).Depending on cell type and stimulus, p38 MAPK can be activatedby MKK3 (17) or MKK6 (27, 46, 52, 61) or by both of them. Insome cell types MKK4 can activate p38 MAPK (17, 44). However,depending upon the stimulus or physiological state, there arecircumstances in which these MKKs can clearly display substratepreferences or noninterchangeable roles (62, 69). For example,MKK3 tends to prefer p38 ${alpha}$ while MKK6 is equally efficient atboth p38 ${alpha}$ and p38ß (18). We also embarked on the currentseries of studies because defining the exact p38 isoform(s)and its immediate activator(s) in the control of UCP1 transcriptionmay provide clear targets to modulate thermogenesis. Using avariety of experimental approaches, we show that p38 ${alpha}$ and itsactivator MKK3 are the sole players in the control of Ucp1 genetranscription.

MATERIALS AND METHODS

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Materials and Methods

Chemicals and plasmids. The plasmid BBCAT, containing the 3.74-kb mouse UCP1 promoterin plasmid pBLCAT6, was a gift from Leslie P. Kozak (38). TheEN-tk-CAT vector containing the UCP1 enhancer (–2530 to–2310), the mouse ß₃AR expression plasmid, andthe ß-actin-luciferase (ß-actin-luc) controlvector for transfections were all previously described (8, 45).The expression vectors pCDNA3-p38 ${alpha}$ and pCDNA3-p38ßMAPK as well as their dominant-negative forms pcDNA3-p38 ${alpha}$ AF andpCDNA3-p38ßAF, all FLAG-tagged, were gifts from JiahuaiHan (26, 32). The mouse pCMV-SPORT6-p38ß MAPK vectorwas purchased from Open Biosystems (Huntsville, AL). A collectionof FLAG-tagged expression vectors created in the plasmid pcDNA3for the following genes were all gifts from Roger J. Davis,University of Massachusetts Medical School: MKK3, MKK4, MKK6,and its constitutively active form, MKK6E (17, 52). The expressionvector pCDNA3-MKK7 was a gift from Josef M. Penninger, Universityof Toronto (70). The S-protein-tagged JLP was a gift from E.Premkumar Reddy, Temple University (5, 40). The full-lengthclones for JIP1, JIP2, and JIP3 were purchased from Open Biosystems(Huntsville, AL) and were subcloned in pcDNA4/HisMax TOPO TAExpression vector from Invitrogen (Carlsbad, Calif.). The ß₃AR-selectiveagonist CL316,243 (CL) was a gift from Elliott Danforth, Jr.(American Cyanamid Co., Pearl River, NY). The anti-FLAG M2-agaroseantibody, dobutamine, forskolin, H89, IGEPAL, isoproterenol,norepinephrine, and salbutamol were from Sigma (St. Louis, MO).Rp-cAMPS was from Biomol Research Laboratory (Plymouth Meeting,PA). SB202190 and SB203580 were from Calbiochem (La Jolla, CA).The nonradioactive PKA activity assay was from Promega (Madison,WI). The Rap1 activation assay was from Upstate (Charlottesville,VA). Specific antibodies to the following epitopes or individualproteins were from Cell Signaling Technologies Inc. (Beverly,MA): 6x-His, p38 MAPK, p38 ${alpha}$ MAPK, phospho-p38 MAPK, MKK3, phospho-MKK3/6,MKK4, phospho-MKK4, MKK7, phospho-MKK7, and glutathione S- transferase(GST)-ATF2. Antibodies specific for the p38 ${alpha}$ and p38ßMAPK isoforms and MKK6 were from Chemicon (Temecula, CA). Forimmunoprecipitation experiments, MKK3, MKK6, and the S-tag antibodieswere obtained from Santa Cruz (Santa Cruz, CA). The S-proteinagarose resin was from Santa Cruz. An additional antibody tophospho-p38 MAPK was from Zymed (South San Francisco, CA). Thesmall interfering RNAs (siRNAs) presented in Table 1 and relatedreagents such as siPORT-Lipid, RNAlater, RNAaquous4PCR, andRNAaquous MIDI RNA extraction kit were from Ambion (Austin,TX). The High Capacity cDNA Archive kit, reverse transcription-PCR(RT-PCR) primers, FAM- and VIC-labeled probes, and the TaqManenzyme were from Applied Biosystems (Foster City, CA). Lipofectamineand precast Tris-glycine polyacrylamide gels were obtained fromInvitrogen (Carlsbad, CA). The alkaline phosphatase-conjugatedsecondary antibodies, the alkaline phosphatase detection reagents,and glutathione Sepharose 4B were from Amersham Biosciences(Piscataway, NJ). Chloramphenicol acetyltransferase (CAT) assays,Complete Protease Inhibitor Cocktail (CPIC) tablets, and proteinG-agarose were from Roche Molecular Biochemicals (Indianapolis,IN). The ProQ Diamond Phosphoprotein Gel Stain and destainingsolution were from Molecular Probes (Eugene, OR). Rosiglitazonewas a gift from GlaxoWellcome Inc. (Research Triangle Park,NC). All other reagents were from the best available sources.

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TABLE 1. Sequences for the siRNAs used in these studies^a

Cell culture and transfection. The HIB-1B brown preadipocytes (57) were maintained in Dulbecco'smodified Eagle medium (DMEM) with 10% fetal bovine serum. Cellsin 6-well plates were transfected with a total amount of plasmidDNA up to 2.2 µg/well and 5 to 10 µl Lipofectamine.As needed, these DNA mixtures included pGL2-ß₃AR (0.5µg), pCDNA3-kinases (0.2 to 1 µg), UCP1 enhancer-TK-CAT(1.0 µg), and ß-actin-luc (0.2 µg) orcytomegalovirus-ß-galactosidase (CMV-ß-GAL)(0.125 µg). Transfection with siRNAs (20 to 60 nM) wereperformed using siPORT lipid (4 to 8 µl). In cotransfectionexperiments involving siRNAs, the siRNA was added to the wellat the time of the seeding the cells, and the plasmid transfectionwas performed 12 h later. In all cases, the PPAR ${gamma}$ agonist rosiglitazone(1 µM) was added at the same time as the serum followingthe serum-free period of the transfection protocols. Where indicated,HIB-1B cells were treated for 1 h with 10 µM H89, 0.5mM Rp-cAMPS, or 5 µM SB prior to treatment with CL (10µM), Forsk (10 µM), dobutamine (10 µM), salbutamol(10 µM), isoproterenol (10 µM), or norepinephrine(1 µM), for which the time of incubation varied in accordto the assay needs. Also, for cells treated with norepinephrine,there was a preincubation phase for 30 min with yohimbine (10µM) and prazosin (1 µM) in order to inhibit anyactivation of ${alpha}$ -adrenergic receptors.

Nonradioactive PKA assay. HIB-1B cells were preincubated for 50 min with H89 (10 µM),Rp-cAMPS (0.1 mM), or SB (5 µM), followed by 10 min with0.2 mM isobutylmethylxanthine in order to inhibit phosphodiesteraseactivity. They were then incubated for 5 min with either CL(10 µM) or Forsk (10 µM). Cells were then washedonce with phosphate-buffered saline (PBS) containing 5 mM ß-glycerophosphateand 1 mM sodium orthovanadate, followed by a lysis buffer (25mM Tris-HEPES, 150 mM NaCl, 10 mM ß-mercaptoethanol,5 mM ß-glycerophosphate, 1 mM sodium orthovanadate,0.5 mM EDTA, 0.5 mM EGTA, 1% triton X-100, 0.1% IGEPAL, and1 CPIC tablet per 10 ml) for 15 min. Five microliters of thiscell lysate was incubated for 30 min at 30°C with 5 µlof the 5x PepTag PKA reaction buffer, 2 µg of PepTag A1peptide (fluorescent kemptide), and 1 ml of the peptide protectionsolution (all parts of the PKA assay kit were from Promega)in a 25-µl total volume. For the positive control, thesample has been replaced by 10 ng of PKA catalytic subunit,and for the negative only lysing buffer is added to the reactionmixture. The reaction was stopped by boiling the sample for10 min, a final concentration of 3.2% glycerol was added, and3.5 µl sample was loaded and resolved on a 0.8% agarosegel. Image acquisition was performed on a typhoon 9410 variablemodes imager and analyzed using ImageQuant TL v2003.03 software.

Rap1 activation assay using RalGDS-RBD. Rap1 pull-down assays were performed essentially as describedpreviously (19) using the reagents from Upstate (Charlottesville,VA). HIB-1B cells were seeded in 10-cm-diameter dishes. Cellswere washed twice in cold PBS and lysed on ice in 1 ml of lysisbuffer (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, 2.5 mM MgCl₂,1% NP-40, 10% glycerol, and 1 Complete Mini Antiprotease tabletper 10 ml of lysis buffer). Cell debris was removed by centrifugationat 13,200 x g for 10 min at 4°C. Fifty microliters of theGST-RalGDS-RBD-agarose slurry was added to each supernatant,and the mixture was incubated at 4°C for 45 min on a rotatingwheel. Beads were washed three times with lysis buffer. Sampleswere denatured for 3 min at 95°C and subjected to sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Western blotting was performed using the anti-Rap1 antibodyincluded in the kit and an alkaline phosphatase-conjugated anti-rabbitsecondary antibody and ECF detection kit from Amersham Biosciences.Image acquisition was performed on a typhoon 9410 variable modesimager and analyzed using imageQuant TL v2003.03 software, bothfrom GE healthcare (Piscataway, NJ).

Protein kinase assay for p38 MAPK activity. HIB-1B cells were transfected or not with FLAG-tagged p38 MAPK,and total cell p38 MAPK activity was assessed from cellularlysate while isoform-specific activity was assessed followingimmunoprecipitation using M2 anti-FLAG agarose antibody. Thecells were washed twice with PBS containing 5 mM ß-glycerophosphateand 1 mM sodium orthovanadate and then lysed for 30 min in a25 mM Tris-HEPES buffer containing 150 mM NaCl, 5 mM ß-glycerophosphate,1 mM sodium orthovanadate, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100,0.1% IGEPAL, and 1 CPIC tablet per 10 ml. Kinase assays in vitrowere performed either on whole-cell lysates or after immunoprecipitation.In the latter case, the lysate was incubated overnight with40 µl of M2 anti-FLAG agarose antibody. Cell lysate orimmune complexes were incubated at 30°C for 45 min with2 µg GST-ATF-2(1-109), 250 µM ATP (containing ornot 10 µCi [ ${gamma}$ -³²P]ATP) in 40 µl of kinase reactionbuffer (43). For the radioactive version of the protocol, anequal amount of 2x Laemmli sample buffer was added to terminatethe reactions. In the nonradioactive version, 40 µl ofglutathione Sepharose 4B was used to pull down the substrate.Proteins were resolved with 4 to 20% acrylamide gradient Tris-glycinegels. Protein phosphorylation was visualized either by autoradiographyfor the radioisotopic protocol or with the Pro-Q Diamond phosphor-proteinstain for the nonradioactive method. In both cases image acquisitionwas performed on a Typhoon 9410 variable-mode imager and analyzedusing ImageQuant TL v2003.03 software.

Western blot for MAPK phosphorylation. MAPK phosphorylation was evaluated by Western blot using specificanti-phospho-MAPK and total MAPK antibodies (1:1,000 dilution)and secondary antibodies (1:10,000 for the Amersham antibodyand 1:25,000 for the Sigma antibodies). The alkaline phosphataseactivity was determined using the ECF detection kit from AmershamBiosciences. Image acquisition was performed on a Typhoon 9410variable modes imager and analyzed using ImageQuant TL v2003.03software.

CAT and luciferase assays. Cells were harvested to assay UCP1 enhancer activities 48 hafter transfection. CL316,243 or Forsk was added for the last6 to 8 h of the transfection to stimulate cAMP production, afterwhich cell extracts were prepared in lysis buffer from a CATenzyme-linked immunosorbent assay (ELISA) kit (Roche MolecularBiochemicals). CAT and luciferase assays were performed as previouslydescribed (8).

RNA isolation, reverse transcription-PCR, and real-time PCR. Total mRNA was extracted from cultured cells using RNAaquous4PCRand from tissue using the RNAaquous MIDI RNA purification kits.For tissues, the samples were submerged in "RNAlater" priorto the extraction. These RNA reagents were from Ambion. cDNAwas generated using the High Capacity cDNA Archive kit fromApplied Biosystems exactly as described in the kit (althoughscaled down to a 50-µl total volume). Real-time PCR wasperformed using TaqMan probes from Applied Biosystems (FosterCity, CA) on an ABI PRISM 7700 Sequence Detector from PerkinElmer (Boston, MA) exactly as indicated by Applied Biosystems.For the quantification of the p38 ${alpha}$ and p38ß MAPK mRNA,standard curves (0 to 4,000 amol) were generated using plasmidscontaining the cDNAs of the mouse genes. GAPDH was used as internalstandard.

Immunoprecipitation. For immunoprecipitation experiments, the tissue or the cellswere lysed with the same buffer as for the kinase assays. Thelysate (1 to 2 mg of total protein) was precleared by a preincubationof 2 h with 40 µl protein G-agarose. The cleared lysatewas then incubated for 3 h with the antibody (kinase assay)or overnight (coimmunoprecipitation) with antibodies. The G-proteinagarose was added, and the mixture was incubated for an additional3 h and washed once with the lysing buffer and five times withthe washing buffer composed of 50 mM Tris-HCl, 150 mM NaCl,and antiproteases. The proteins are eluted from the resin inan ultrafree-mc 5-µm centrifugal filtration device byexposing the resin to sample buffer without reducing agent atroom temperature for 10 min.

RESULTS

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Results

In previous studies we demonstrated that p38 MAPK activity isinvolved in the ßAR- and cAMP-dependent inductionof the Ucp1 gene in brown adipocytes (7, 8). However, neitherthe identity of the p38 MAPK nor the molecular intermediarieslinking PKA to the activation of p38 MAPK are known. Therefore,we used a series of experiments designed to define the p38 MAPKisoforms and immediate upstream activators involved. In ourearlier studies, we presumed that the elevated cAMP levels generatedin response to ß-agonist stimulation are activatingPKA, concluding that this kinase is solely responsible for conveyingthe cAMP signal that leads to p38 MAPK activation and Ucp1 geneexpression. This conclusion was based on the ability of twomechanistically different "inhibitors" of cAMP, the competitiveantagonist Rp-cAMPS and the catalytic inhibitor H89, to suppressboth p38 MAPK activation and transcription of the Ucp1 gene.This "signature" typically indicates involvement of PKA. However,in a variety of cell types, cAMP has been shown to activatethe small G-protein Rap1 through its interaction with a familyof guanine nucleotide exchange factors (GEFs) that include Epac(exchange protein directly activated by cAMP), cAMP-GEF-I, andcAMP-GEF-II (1, 33, 65). The activities of these molecules areblocked by Rp-cAMPS but are unaffected by H89. Importantly,Rap1 has been shown to be an activator of p38 MAPK (30, 59).Therefore, as we began this series of studies it was necessaryto unequivocally determine whether PKA or a GEF (or some combinationof both) leads to stimulation of p38 MAPK and Ucp1 gene expression.We treated HIB-1B brown adipocytes with the ß₃AR agonist,CL316,243 (CL), or the adenylyl cyclase stimulator, forskolin(Forsk), in the presence of H89 or Rp-cAMPS or the p38 MAPKinhibitor SB202190 (SB). As shown in Fig. 1A, PKA was activatedby either CL or Forsk. The response to both activators was blockedby H89 or Rp-cAMPS but not by SB. Therefore, these results indicateactivation of PKA and additionally show that inhibition of p38MAPK does not affect PKA. As shown in Fig. 1B, neither CL norForsk was able to elicit GTP loading to Rap1. Together theseresults strongly support the conclusion that p38 MAPK activationby cAMP does not depend upon a cAMP-GEF and Rap1 activationbut, rather, solely requires PKA.

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FIG. 1. ß-Adrenergic receptor (ßAR) agonists or Forskolin (Forsk) promotes uncoupling protein 1 (UCP1) enhancer activity through protein kinase A (PKA) and p38 MAPK. HIB-1B cells were transfected (A to D) or not (E and F) with the mouse ß₃AR. (A to D) Cells were preincubated 1 h with the solvent only (Cont), 10 µM H89, 0.5 mM Rp-cAMPS, or 5 µM SB202190, and then the cells were incubated with the solvent only (Basal), 10 µM CL316,243 (CL), or 10 µM Forskolin (Forsk). (A) After 5 min, the cells were lysed and PKA activity was measured in the whole-cell lysate using kemptide as substrate. (B) Also after 5 min, Rap1 activation was evaluated using a Ral-GDS pull-down assay. (C) After 20 min, p38 MAPK activity was measured using GST-ATF2 as substrate. (D) After 6 h, UCP1 enhancer activity was evaluated using a UCP1 enhancer-chloramphenicol acetyltransferase (CAT) assay: Basal (white bars), CL (oblique bars), and Forsk (black bars). (E and F) Cells were preincubated 1 h with the solvent only (Cont), 10 µM H89, or 5 µM SB202190, and then the cells were preincubated an additional 15 min with 10 µM yohimbine and 1 µM prazosin (for the norepinephrine treated cells only) or the solvent, and then the cells were incubated with the solvent only (Basal), 10 µM dobutamine (Dobu), 10 µM salbutamol (Salbu), 10 µM isoproterenol (Iso), or 1 µM norepinephrine (NE). (E) After 20 min, p38 MAPK activity was measured using GST-ATF2 as substrate. (F) After 6 h, UCP1 enhancer activity was evaluated by CAT assays: Basal (white bars), Dobu (horizontal bars), Salbu (oblique bars), Iso (black bars), and NE (reverse oblique bars). The blots shown are the results of one of two (E), three (A and B), or five (C) experiments. The results presented in graphs are the means ± standard deviations of two (F) or three (D) independent experiments, each performed in duplicate.

To confirm the role of both PKA and p38 MAPK in UCP1 induction,HIB-1B cells were pretreated with H89, Rp-cAMPS, or SB, followedby stimulation with either CL or Forsk. Activation of p38 MAPKwas measured using glutathione-S-transferase (GST)-tagged ATF-2as a substrate. As shown in Fig. 1C, p38 MAPK enzyme activitystimulated by CL or Forsk was abrogated by H89, Rp-cAMPS, andSB. These inhibitors similarly blocked the transactivation ofthe UPC1 enhancer in response to CL and Forsk (Fig. 1D). Theseresults indicate that, irrespective of the stimulus, PKA isnecessary for p38 activation and that, in turn, induction ofUCP1 enhancer activity requires p38 MAPK activity.

Since all three ßARs are expressed in brown adipocytesand can stimulate cAMP production (56), we proposed that allof them can activate p38 MAPK and UCP1 transcription. To testthis hypothesis, we treated HIB-1B cells with specific agonistsof these receptors. As shown in Fig. 1E, the ß₁AR-selectiveagonist, dobutamine, and the ß₂AR-selective agonist,salbutamol, both activated p38 MAPK in a PKA-dependent manner.The nonselective ßAR activator isoproterenol and thenatural adrenergic agonist norepinephrine also activated p38MAPK in a PKA-dependent fashion (Fig. 1E). Furthermore, thesefour ßAR agonists also induced UCP1 enhancer activation,which was blocked by p38 MAPK inhibition (Fig. 1F). These resultsshow that all three ßAR subtypes can stimulate p38MAPK activity and subsequently Ucp1 gene transcription.

To determine whether ß₃AR stimulation leads to p38MAPK activation and to a p38 MAPK-dependent Ucp1 gene expressionin brown fat in vivo, SB (12.5 mg/kg of body weight) and CL(1 mg/kg) were administered to mice. Phosphorylation and activationof p38 MAPK and JNK was assessed by Western blotting and kinaseassays and Ucp1 gene expression by real-time PCR. As shown inFig. 2A, CL treatment induced phosphorylation of p38 MAPK by2.5- ± 0.2-fold and p38 MAPK enzyme activity by 2.4-± 0.1-fold. In contrast, following CL treatment, phosphorylationof JNK could not be detected (Fig. 2B). The ability of antibodyto recognize phospho-JNK was confirmed by treating HIB-1B cellswith 5 µg/ml anisomycin for 15 min (Fig. 2B). Under thesesame treatment conditions, CL injection stimulated Ucp1 geneexpression, and this stimulation was largely prevented (70%)by prior p38 MAPK inhibition (Fig. 2C). Consistent with whatwe have previously reported (7), these results clearly showthat selective ß₃AR agonist stimulation in vivo triggersp38 MAPK activity to regulation of Ucp1 gene transcription.

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FIG. 2. ß₃AR agonist CL316,243 promotes uncoupling protein 1 (Ucp1) gene expression in vivo through p38 MAPK. Mice were pretreated with two injections of saline (Control) or 12.5 mg/kg SB203580. The first injection was 25 h and the second 1 h prior to the saline (Basal) or CL injection (1 mg/kg). (A) After 30 min, the brown adipose p38 MAPK activation was evaluated by Western blotting using an anti-phospho-p38 MAPK antibody (top blot) or using GST-ATF2 as a substrate (middle blot). (B) After 30 min, JNK activation was evaluated by Western blot. (C) After 6 h, Ucp1 gene expression was evaluated by real-time PCR (triplicate samples per group) using Taqman probe: Basal (white bars) and CL (gray bars). The blots shown are the results of one of two experiments using two mice per each treatment group. For panels A and B, HIB-1B cells treated with anisomycin (5 µg/ml) were used as a positive control. The results presented in graphs are the means ± standard deviations of two independent experiments using two mice per treatment group.

To identify the p38 MAPK isoforms(s) responsible for UCP1 enhanceractivation, we first assessed which SB-sensitive isoforms wereexpressed in BAT and in the brown adipocyte cell line used todissect the molecular pathway between PKA and Ucp1 gene expression.As shown in Fig. 3A, both p38 ${alpha}$ and -ß mRNAs were expressedin BAT as well as in HIB-1B cells. Consistent with this finding,both proteins were detected by Western blot (Fig. 3B). We nextoverexpressed the p38 ${alpha}$ or -ß isoforms in HIB-1B cellsand measured UCP1 promoter activity. As shown in Fig. 3C, bothisoforms could stimulate UCP1 enhancer activation equally (witha slight preference for the ${alpha}$ isoform). Next, we coexpressedMKK6E (a constitutively active form of this kinase that canphosphorylate and activate p38 MAPK) with either p38 ${alpha}$ or -ßin HIB-1B cells, followed by measurements of UCP1 enhancer activity.As shown in Fig. 3D, MKK6E could activate either of the p38isoforms as measured by significant amplification of UCP1 enhanceractivity, but there was a greater preference for p38 ${alpha}$ MAPK. Together,these results indicate that both p38 ${alpha}$ and -ß isoformsare capable of stimulating UCP1 transcription and that underconditions of maximal stimulation p38 ${alpha}$ MAPK might couple moreefficiently to UCP1 induction. However, these data do not indicatewhether either or both isoforms play a role under adrenergicstimulation. To address this issue, we introduced FLAG-taggedp38 ${alpha}$ or -ß MAPK into HIB-1B cells and subsequentlytreated the cells with CL or Forsk. As clearly shown in Fig.4A and B, p38 ${alpha}$ MAPK but not p38ß was activated by CLor Forsk. We also performed immunoprecipitation experimentsof the endogenous p38 MAPK isoforms and confirmed that Forsk-inducedp38 MAPK activity could be recovered only from the p38 ${alpha}$ MAPKimmunoprecipitate (Fig. 4C). Interestingly, using our brownadipocyte model, transactivation of the UCP1 enhancer by CLor Forsk was potentiated only by p38 ${alpha}$ but not by the p38ßisoform (Fig. 4D). In order to validate this selectivity invivo, mice were either injected with 1 mg/kg CL or exposed toa 4°C environment. As shown in Fig. 4E and F, both manipulationsled to the sole activation of the p38 ${alpha}$ MAPK isoform in interscapularBAT. Altogether, these data establish that p38 ${alpha}$ MAPK but notp38ß is activated during sympathetic nervous systemstimulation of the thermogenic program and following exposureof BAT and brown adipocytes to sympathomimetic drugs.

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FIG. 3. p38 ${alpha}$ or p38ß MAPK can promote UCP1 enhancer activity. The expression of p38 ${alpha}$ and p38ß MAPKs was measured in BAT and HIB-1B cells by quantitative real-time PCR (A) and Western blotting with selective antibodies for each isoform (B). (C) HIB-1B cells were transfected with increasing concentration of FLAG-tagged p38 ${alpha}$ MAPK. Two days later, the kinases were immunoprecipitated and their expression was evaluated by Western blot using anti-p38 MAPK antibody (second and fourth blots); we measured their activity using GST-ATF2 as a substrate (first and third blots), and we evaluated UCP1 enhancer activity using a CAT assay (graph). (D) HIB-1B cells were transfected with FLAG-tagged p38 MAPK and/or FLAG-tagged MKK6E. Two days later, theirs kinases were immunoprecipitated and their expression was measured by Western blot using anti-p38 MAPK and anti-MKK6 antibodies (second, third, and fourth blots), p38 MAPK activity was measured using GST-ATF2 as substrate (first blot), and UCP1 enhancer activity was evaluated by CAT assay (graph). The results shown are means ± standard deviations of three independent experiments, each performed in triplicate, while the blots are from one of three experiments.

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FIG. 4. p38 ${alpha}$ MAPK is selectively activated following ß-adrenergic or forskolin stimulation. (A and B) HIB-1B cells were transfected with the ß₃AR and with FLAG-tagged p38 ${alpha}$ MAPK (A) or FLAG-tagged p38ß MAPK (B). (A and B) Cells were treated as detailed in Fig. 1A. Cells were lysed, the kinase was immunoprecipitated (Western blot, bottom blots in A and B), and activity was measured using GST-ATF2 as a substrate (top blots in A and B). The results shown are from one of three independent experiments. (C) HIB-1B cells were treated as shown, and p38 ${alpha}$ and p38ß MAPKs were immunoprecipitated and kinase activity measured using GST-ATF2 as a substrate. (D) HIB-1B cells were transfected with the ß₃AR and with FLAG-tagged p38 ${alpha}$ MAPK or FLAG-tagged p38ß MAPK. Cells were treated as follows: Basal (white bars), CL (gray bars), and Forsk (black bar). For measurement of UCP1 enhancer activity cells were harvested after 6 h. The results shown are means ± standard deviations of three independent experiments, each performed in duplicate. For measurement of kinase activity, 20 min posttreatment the kinases were immunoprecipitated and activity was measured using GST-ATF2 as a substrate (upper blot). Relative amounts of the kinases in the assay are shown by Western blot using anti-FLAG antibody (lower blot). (E and F) Mice were either treated with CL (1 mg/kg intraperitoneally) for 30 min (E) or placed at 4°C for 1 h (F), and BAT was excised and processed for immunoprecipitation of p38 ${alpha}$ MAPK or p38ß MAPK. Kinase activity and the protein levels of the p38 MAPK isoforms was measured as above.

Establishing that the ${alpha}$ isoform of p38 MAPK is the one that isactivated, we used siRNA gene silencing to demonstrate thatp38 ${alpha}$ MAPK and not p38ß MAPK was responsible for theinduction of UCP1 expression. In these studies it was firstnecessary to demonstrate the efficacy of the siRNAs directedagainst either the p38 ${alpha}$ or -ß isoforms. This was examinedin HIB-1B cells. As shown in Fig. 5A, the siRNA against eitherform of p38 MAPK reduced the targeted protein level by morethan 80% without affecting the other isoform. More importantly,as shown in Fig. 5B, using this approach we found that essentiallyall CL- and Forsk-promoted p38 activity can be attributed tothe p38 ${alpha}$ MAPK isoform. Figure 5C further shows that the siRNAagainst p38 ${alpha}$ MAPK completely inhibited UCP1 enhancer activation,while the siRNA against p38ß failed to do so. Similarresults were obtained in experiments employing dominant-negativeconstructs of p38 ${alpha}$ and p38ß MAPK (not shown). Finally,it was rather remarkable to find that even the 60- to 80-foldinduction of the endogenous Ucp1 gene in HIB-1B cells was totallyeliminated by the p38 ${alpha}$ MAPK siRNA (Fig. 5C). Altogether thesedata leave little doubt about the highly specific activationof p38 ${alpha}$ MAPK by ßAR agonists and PKA and its essentialrole in the activation of the Ucp1 gene.

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FIG. 5. p38 ${alpha}$ MAPK is essential for ß₃AR agonist or Forsk induction of Ucp1 gene expression. (A and B) HIB-1B cells were transfected with siRNA targeting p38 ${alpha}$ and p38ß MAPK, and 48 h later kinases were immunoprecipitated with specific anti-p38 ${alpha}$ MAPK (top blot) or p38ß MAPK (bottom blot). (A) Presence of the kinases was measured by Western blot using isoform nonselective p38 MAPK antibody (both blots). The results shown are from one of two independent experiments. (B) Kinase activity was measured using GST-ATF2 as a substrate. (C) HIB-1B cells were transfected with siRNA targeting p38 ${alpha}$ and p38ß MAPK and 12 h later with the ß₃AR, UCP1 enhancer, and ß-actin-luciferase plasmids. Forty-eight hours later, the cells were incubated with the solvent (Basal), and 10 µM CL or 10 µM Forsk was used for 6 h for the measurement UCP1 enhancer activity a CAT reporter system: Basal (white bars), CL (gray bars), Forsk (black bars). The results shown are means ± standard deviations of two independent experiments, each performed in triplicate. (D) HIB-1B cells were transfected with siRNA targeting p38 ${alpha}$ and p38ß MAPK and expression vector for ß₃AR. The cells were incubated with the solvent (Basal), 10 µM CL, or 10 µM Forsk for 6 h, the RNA was extracted, and the measurement of UCP1 was evaluated by real-time PCR using a TaqMan probe: Basal (white bars), CL (gray bars), and Fork (black bars). The results shown are means ± standard deviations of two determinations, each performed in triplicate.

The next objective was to address the origin of p38 ${alpha}$ MAPK selectivity.For this purpose we explored which of the MKKs were activatedby CL and Forsk. In Fig. 6A (middle panel), MKK3 and/or MKK6was phosphorylated in a PKA-dependent manner following CL orForsk. However, neither CL nor Forsk was able to promote thephosphorylation of MKK4 or MKK7 (Fig. 6B). These results areconsistent with the fact that the JNK pathway is not activatedin brown adipose tissue in vivo (Fig. 3B) or in brown adipocytesin vitro (Fig. 6B). However, the nonisoform selective natureof the MKK3/6 phospho-antibody required additional experimentationin order to address whether one (or both) of these two MKK isoformswas activated. We performed selective immunoprecipitation ofMKK3 and MKK6 under basal and cAMP-stimulated conditions. Asshown in Fig. 6C, Forsk-induced phospho-MKK3/6 immunoreactivitywas detected only upon immunoprecipitation of MKK3. Confirmationof this exclusive activation of MKK3 versus MKK6 in vivo wasobtained from BAT samples of mice treated with CL or placedin a 4°C environment. Collectively, these findings demonstratea selective activation at the level of the direct upstream kinasewithin the p38 MAPK module, which might be an underlying mechanismof the above-described specific p38 ${alpha}$ MAPK activation.

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FIG. 6. ß-Adrenergic agonists and cAMP promotes activation of MKK3. (A and B) HIB-1B cells were treated as described in Fig. 1A and then lysed for measurement of the activity and amounts of the indicated kinases by Western blotting as detailed in Materials and Methods, and MKK3/6 phosphorylation was evaluated by Western blot (middle blot). Each blot shown is one of three experiments. (C) HIB-1B cells were treated or not with H89 and forskolin as described in Fig. 1A. The cells were lysed, MKK3 and MKK6 were immunoprecipitated, and their phosphorylation state was measured by Western blotting. (D and E) Mice were either treated with CL (1 mg/kg intraperitoneally) for 30 min (E) or placed at 4°C for 1 h (F), and BAT was excised and processed for immunoprecipitation of MKK3 or MKK6. The ability to phosphorylate GST-tagged p38 ${alpha}$ MAPK and the protein levels of the MKKs were measured by Western blotting.

MKK6 is a universal p38 MAPK activator, but the ability of MKK3to activate p38ß MAPK is modest at best (18). It wastempting to speculate that the specific involvement of p38 ${alpha}$ MAPKwould be recapitulated at the level of MKK3. We therefore testedthe functional impact of overexpressing MKK3, MKK4, MKK6, andMKK7 individually on UCP1 enhancer activity and p38 MAPK activity.The results clearly show that MKK3, and to a much lower extentMKK6, can potentiate the effects of CL and Forsk on both UCP1expression (Fig. 7A) and p38 MAPK activity (Fig. 7B). The reciprocalexperiments, using siRNAs that specifically downregulated MKK3and MKK6 by more than 80% (Fig. 7C), show that only the selectivesiRNA directed against MKK3 could block CL and Forsk inductionof p38 MAPK activity (Fig. 7D) as well as UCP1 expression (Fig.7E). Finally, the siRNA that specifically targets MKK3 was theonly one capable of completely interfering with the expressionof the endogenous Ucp1 gene (Fig. 7F). All together, these resultsunequivocally define the proximal steps in the cAMP- and PKA-dependentactivation of the Ucp1 gene in brown fat as being mediated solelyby MKK3 and p38 ${alpha}$ MAPK.

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FIG. 7. MKK3 is essential for ß₃AR agonist or Forsk induction of Ucp1 gene expression. (A) HIB-1B cells were transfected with the ß₃AR, with FLAG-tagged MKK3, MKK4, MKK6, or MKK7, and with the enhancer-CAT plasmid. Two days later the cells were treated with the solvent (Basal), 10 µM CL, or 10 µM Forsk. The treatment was stopped after 6 h for the measurement UCP1 enhancer activity in a CAT reporter system: Basal (white bars), CL (gray bars), and Forsk (black bars). (B) HIB-1B cells were transfected with siRNA targeting MKK3 or MKK6, and 2 days later its expression was evaluated by Western blots. (C) HIB-1B cells were transfected with siRNA targeting MKK3 or MKK6 and 12 h later with the ß₃AR, the UCP1 enhancer, and the ß-actin-luciferase plasmids. Forty hours later, the cells were incubated with the solvent (Basal), 10 µM CL, or 10 µM Forsk for 6 h for the measurement UCP1 enhancer activity in a CAT reporter system: Basal (white bars) and CL (gray bars). (D) HIB-1B cells were transfected with siRNA targeting p38 ${alpha}$ and p38ß MAPK and 12 h later with the ß₃AR. The cells were incubated with the solvent (Basal), 10 µM CL, or 10 µM Forsk for 6 h, the RNA was extracted, and the measurement of UCP1 was evaluated by real-time PCR using a TaqMan probe. The results shown are means ± standard deviations of two determinations, each performed in triplicate.

The MAP kinases are usually assembled together with their upstreamMKKs into a signaling module that is coordinated by large scaffoldingmolecules such as the JIPs (JNK-interacting proteins) (21, 47).As a result, these scaffold proteins can concentrate interactingsignaling partners in the vicinity of an upstream activatorin order to favor a particular pathway. Since there is no establishedlink between PKA and p38 MAPK, we attempted to determine which,if any, of the known JIP family members in brown adipocytesmight serve as the scaffold to specifically bind p38 ${alpha}$ MAPK andMKK3. The first objective was to determine the relative expressionlevels of the four known JIPs: JIP1, JIP2, JIP3, and JLP. Asshown in Fig. 8A, there was no detectable expression of JIP1in either BAT or the HIB-1B cell line (RT-PCR cycle, >45).However, JIP2, JIP3, and JLP were all found in both samples,with JIP2 being the least abundant at the mRNA level (Fig. 8A).Based on these results, each of these three JIPs was analyzedfor its ability to interact specifically with MKK3 and p38 ${alpha}$ MAPK.His-tagged or S-protein-tagged constructs of JIP2, JIP3, andJLP were transfected into HIB-1B cells, followed by their immunoprecipitationin order to assess the identity of any interacting kinases.Figure 8B clearly shows that only JIP2 was able to specificallyrecover both MKK3 and p38 ${alpha}$ MAPK. It is noteworthy that neitherMKK6 nor p38ß MAPK was found under any circumstance,although all these molecules are clearly present as shown inthe lysate.

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FIG. 8. p38 ${alpha}$ MAPK and MKK3 are present in a complex with JIP2. The identification and quantification of the JIPs at the mRNA level was performed by quantitative real-time PCR (A). JIP1 was undetectable in these assays (not shown). (B) HIB-1B cells were transfected with pcDNA3 (first lane), His-tagged JIP2 (second lane), His-tagged JIP3 (third lane), or S-protein-tagged JLP (fourth lane). Tagged proteins were immunoprecipitated (top two gels), and MKK3, MKK6, p38 ${alpha}$ , and p38ß MAPK were detected by Western blotting in the recovered immunoprecipitate (upper panel) and the lysate (bottom panel).

DISCUSSION

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Discussion

Catecholamine regulation of brown fat thermogenesis has beenclearly shown to involve increased transcription of the Ucp1gene as a result of stimulation of the ßARs and cAMPproduction (see references 48 and 51 for reviews). Despite this,the details of the signaling cascade beyond this point havebeen ambiguous. It has been generally assumed that the elevatedlevels of cAMP lead to activation of PKA, which in turn phosphorylatesthe nuclear factor CREB, resulting in transcription of the varioustarget genes in brown fat, including UCP1. In the last few yearswe have reported results from studies in white and brown adipocytecell models and in vivo manipulations to show that p38 MAPKis activated in response to ß-adrenergic stimuli (7,8). This was a novel and unexpected link between cAMP and p38MAPK, but one that relied heavily on the use of chemical inhibitorsof p38 MAPK and PKA. Since there is no clearly delineated seriesof steps linking PKA to p38 MAPK in the literature, unequivocalidentification of the individual molecules in this cascade requiresthe use of more stringent approaches.

The p38 MAPK group is composed of four isoforms, p38 ${alpha}$ (26, 41),p38ß (32), p38 ${gamma}$ (43), and p38 ${delta}$ (66). Among them, p38 ${alpha}$ and -ß are sensitive to the pyrimidyl imidazoles SB202190and SB205380 (14, 23). Our earlier reports indicated that ßARand PKA stimulation of UCP1 expression in brown adipocytes wassensitive to SB (7, 8) and thus narrowed the scope of inquiry.The pyridinyl imidazole-sensitive p38 MAPK isoforms are oftenconsidered to be redundant enzymes, as their substrate specificitiesoverlap significantly. However, in some cases these two isoformshave been shown to be able to discriminate between substrates,at least under conditions of forced overexpression of dominant-negativemutants (67, 73).

In the studies that we report here, we used a combination ofin vivo and in vitro approaches designed to distinguish betweenthe p38 MAPK isoforms and to identify the upstream MKK enzyme(s)responsible for mediating the PKA signal to activate Ucp1 geneexpression. Our results clearly establish that p38 ${alpha}$ MAPK is acentral obligatory component of this signaling cascade, witharguably no contribution from p38ß MAPK, despite itspresence. We do not at this point rule out the possibility thatp38ß MAPK might regulate other genes in the brownadipocyte that might be triggered by different stimuli. Forexample, insulin has been reported to selectively activate p38ßMAPK but not p38 ${alpha}$ in brown adipocytes, and this effect was associatedwith increased glucose transport (36).

Our studies also indicate that there is a unique requirementfor MKK3 as the immediate upstream kinase for p38 ${alpha}$ . The selectivityof these two elements to be activated in succession is not particularlysurprising, but nevertheless it does occur in spite of the existencein brown adipocytes of at least four MKKs, three of which havebeen demonstrated to activate p38 ${alpha}$ in other settings (17, 27,44, 46, 52, 61). This tight coupling is most likely indicativeof their existence in a multimolecular signaling complex, asis known to exist for the stress-activated kinases from yeastto mammals (21, 47). These signaling modules are maintainedtogether by scaffolding proteins, such as KSR for the ERK pathwayand the JIP proteins for the JNK pathway. A recent estimateof the size of this "functional family" of scaffold proteinssuggests more than 19 members (47). Clearly, this complexitynecessitates more extensive investigation in order to assignindividual scaffolds to specific kinase members but which willprobably also depend upon the stimuli and the cell types inwhich they exist. Much is known about the role of these JIPscaffolding molecules for the JNK pathway (for which they werenamed), while in the case of p38 MAPK three members of the family(JIP2, JLP, and JIP4) have been proposed to be able to interactwith p38 MAPK and/or MKK3 (5, 34, 40). When considering adipocytesit must be acknowledged that relatively little is known aboutthese molecules aside from a recent report concerning a rolefor JIP1 in insulin-induced JNK activation (31). Nevertheless,since we have now established that PKA specifically utilizesMKK3 and p38 ${alpha}$ MAPK to regulate genes in the brown adipocyte,this information directed our quest for the scaffolding proteininvolved. Our results clearly show that at least three putativep38 MAPK scaffolds are expressed in brown adipose tissue. Theabsence of JIP1 and the presence of JIP2 was somewhat surprising,since JIP1 is clearly expressed at low levels in white adiposetissue (31) and JIP2 is expressed predominantly in neuronaltissue (71). However, as usual, adipose tissue was absent fromthe panel of tissue surveyed for JIP2 expression in this latterstudy. Since JIP3 and JLP are more widely expressed, their existencein adipose tissue was not unexpected. Interestingly, when JIP2was immunoprecipitated, only MKK3 and p38 ${alpha}$ MAPK were recoveredwithin the coprecipitate. Although not a definitive proof ofthe necessity of JIP2 for the PKA-dependent activation of p38 ${alpha}$ MAPK, it at least places these components within close proximityto each other. Also, during the revision of the manuscript JIP4was cloned (34), therefore the role for JIP4 in parallel withJIP2 cannot be excluded.

From a signaling perspective, the adipocyte is unique in thatall three known ßAR subtypes are expressed there andeach is coupled to the production of cAMP (56). Here we havealso shown that all three ßARs can stimulate p38 MAPKin the adipocyte to increase Ucp1 gene expression. Based onthe present results and our earlier findings that cAMP-dependentactivation of p38 MAPK elicits an orchestrated response to increasethe thermogenic capacity of brown adipocytes by increasing theexpression of PGC-1 ${alpha}$ , a master regulator of mitochondriogenesis(68), an interesting speculation arises. Since human adipocytesalso express multiple ßAR subtypes and the criticalregion of the Ucp1 gene that responds to cAMP and p38 MAPK isconserved between rodents and humans, there may well be a similarregulation of p38 MAPK and activation of UCP1 and PGC-1 ${alpha}$ expressionin human adipocytes. This prospect will require careful examinationfrom human visceral adipose samples, since this depot is themain location of brown adipocytes in adult humans and may potentiallybe a reservoir of quiescent cells capable of thermogenic activityupon appropriate stimulation.

ACKNOWLEDGMENTS

We thank Leslie P. Kozak, Jiahuai Han, Roger J. Davis, JosefM. Penninger, and E. Premkumar Reddy for their invaluable giftsof plasmids (listed in Materials and Methods). We also thankAlexander Medvedev for helpful discussions in the initial phaseof the project.

This work was supported by NIH awards R01 DK57698 and R01 DK53092(S.C.) and a fellowship from Fonds de la Recherche en Santédu Québec (J.R.).

FOOTNOTES

* Corresponding author. Mailing address: CIIT Centers for Health Research, 6 Davis Drive, Box 12137, Research Triangle Park, NC 27709. Phone: (919) 558-1378. Fax: (919) 558-1305. E-mail: scollins{at}ciit.org.

REFERENCES

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