p38 mitogen-activated protein kinase is the central regulator of cyclic AMP-dependent transcription of the brown fat uncoupling protein 1 gene

Abstract

It is well established that catecholamine-stimulated thermogenesis in brown fat requires beta-adrenergic elevations in cyclic AMP (cAMP) to increase expression of the uncoupling protein 1 (UCP1) gene. However, little is known about the downstream components of the signaling cascade or the relevant transcription factor targets thereof. Here we demonstrate that cAMP- and protein kinase A-dependent activation of p38 mitogen-activated protein kinase (MAPK) in brown adipocytes is an indispensable step in the transcription of the UCP1 gene in mice. By phosphorylating activating transcription factor 2 (ATF-2) and peroxisome proliferator-activated receptor gamma (PPARgamma) coativator 1alpha (PGC-1alpha), members of two distinct nuclear factor families, p38 MAPK controls the expression of the UCP1 gene through their respective interactions with a cAMP response element and a PPAR response element that both reside within a critical enhancer motif of the UCP1 gene. Activation of ATF-2 by p38 MAPK additionally serves as the cAMP sensor that increases expression of the PGC-1alpha gene itself in brown adipose tissue. In conclusion, our findings illustrate that by orchestrating the activity of multiple transcription factors, p38 MAPK is a central mediator of the cAMP signaling mechanism of brown fat that promotes thermogenesis.

FIG. 1.

Cold exposure promotes p38 MAPK phosphorylation, PGC-1α expression, and UCP1 transcription in IBAT. As described in Materials and Methods, IBAT was harvested from individual mice exposed to the cold to measure the following factors. (A) Phosphorylated p38 MAPK (p38-P) and total p38 MAPK (p38-T) in IBAT, measured by immunoblotting with specific antisera. (B) PGC-1α and UCP1 mRNA detected by Northern blotting. (C) PGC-1α protein assessed by immunoblotting with specific antisera; each lane represents the average of three mice per group. The results shown are from one of four independent experiments.

FIG. 2.

Cold activation of p38 MAPK is necessary for UCP1 expression in IBAT. As described in Materials and Methods, mice were treated with two doses of the p38 MAPK inhibitor SB203580 (+SB) or vehicle solution (−SB) at 16 and 1 h before the cold exposure. IBAT was harvested from each animal to measure the levels of p38 MAPK activity and UCP1 mRNA. (A) p38 MAPK activity measured by in vitro phosphorylation of the p38 MAPK specific substrate, the ATF-2 fragment, by IBAT lysates. Levels of p38 MAPK in the tissue lysates were measured by immunoblotting with antibodies against p38 MAPK. (B) UCP1 mRNA detected by Northern blotting. The level of 18S RNA was detected with methylene blue in the blot. All results are from one of four independent experiments.

FIG. 3.

(A) Both PPRE and CRE2 of the UCP1 enhancer are necessary for p38 MAPK-dependent activation of the UCP1 enhancer. As detailed in Materials and Methods, HIB-1B cells were transfected with wild-type UCP1 enhancer (EN-Tk-CAT) or the UCP1 enhancer containing a point mutation at either PPRE (PPRE^mut) or CRE2 (CRE2^mut). As indicated, cells were either treated with FSK (10 μM) during the last 6 h of the transfection or cotransfected with an expression vector for dominant negative p38 MAPKα (p38αAF) or constitutively active MKK6 (MKK6E). The CAT activities were measured and normalized to β-actin luciferase. The results shown are means ± standard deviations of three independent experiments, each performed in duplicate. (B) p38 MAPK regulates the activities of the consensus PPRE and CRE in brown fat cells. The [AOX]₂-tk CAT plasmid containing two copies of consensus PPRE or CRE (α18CRE) was introduced into HIB-1B cells via transient transfection. Cells were then treated with FSK for the last 6 h of the transfection in the absence or presence of incubation with SB. CAT activities were measured and normalized to β-actin luciferase. The results shown are means ± standard deviations of two independent experiments. (C) Both ATF-2 and PPARγ, but not CREB, bind to the UCP1 enhancer. HIB-1B cells were differentiated for 5 days as detailed in Materials and Methods. Cells were then stimulated with FSK for 6 h, followed by incubation with 1% formaldehyde to cross-link DNA and transcriptional factors and histones. DNA fragments sheared by sonication were precipitated with antibodies against ATF-2, CREB, or PPARγ, and the presence of the UCP1 enhancer and the proximal region of the UCP1 promoter containing the CRE4 in the precipitates was detected by PCR with specific primers. Results shown are representative of three independent experiments.

FIG. 4.

Phosphorylation of PGC-1α by p38 MAPK is necessary for cAMP-dependent UCP1 expression. (A) UCP1 enhancer activity reconstituted in COS-7 cells. COS-7 cells were cotransfected with EN-Tk-CAT and expression vectors of PGC-1α (0.5 μg/well), PPARγ (0.25 μg/well), and RXRα (0.25 μg/well). Rosiglitazone (1 μM) (Rosi) was added to the cells at the beginning of the transfection as noted. Cells were treated with FSK (10 μM) during the last 6 h of transfection. In panels B and C, COS-7 cells were similarly cotransfected with EN-Tk-CAT and the expression vectors of PPARγ, RXRα, and various amounts of wild-type PGC-1α. As indicated, some cells were treated with FSK with or without a preincubation with SB (B) or the PGC-1α, mutant PGC-1αA3 (C). The white bar in panel A represents the UCP1 enhancer activity in the absence of PPARγ/RXRα, rosiglitazone, PGC-1α, and FSK. In the experiments described above, CAT activities were measured in whole-cell lysates 48 h after the transfection and were normalized to luciferase. The results shown are means ± standard deviations of three independent experiments, each performed in duplicate. (D) PGC-1α stimulation of UCP1 gene transcription requires its phosphorylation by p38 MAPK in primary brown adipocytes. Cells were prepared as described in Materials and Methods, infected with empty retrovirus vector (V) or the vectors expressing wild-type pMSCV-PGC-1α (Wt) or the PGC-1α mutant pMSCV-PGC-1αA3 (Mut), and stimulated with βAR agonist ISO (10 μM) for 6 h. The transcripts of UCP1 and cyclophilin (Cyclo) were detected by Northern blotting. The results shown are means ± standard deviations of three independent experiments.

FIG. 5.

p38 MAPK is necessary for PGC-1α expression and differentially regulates the phosphorylation of CREB and ATF-2 in IBAT. As detailed in Materials and Methods, mice pretreated with the p38 MAPK inhibitor SB (+SB) or vehicle (−SB) were placed at 4°C for the time indicated. IBAT was harvested from individual mice to analyze the following factors by Northern or Western blotting. (A) PGC-1α mRNA and 18S RNA (same as in Fig. 2B). (B) PGC-1α protein. (C) Phosphorylated CREB (CREB-P) and total CREB (CREB-T). (D) Phosphorylated ATF-2 (ATF-2-P) and total ATF-2 (ATF-2-T). The results represent one of four independent experiments.

FIG. 6.

cAMP-dependent PGC-1α transcription in brown adipocytes is regulated by p38 MAPK. (A) Effect of the PKA inhibitor (H89) and p38 MAPK inhibitor (SB) on β-adrenergic stimulation of PGC-1α expression in primary brown adipocytes. Primary brown adipocytes were isolated and differentiated as described in Materials and Methods. Following incubation with CL (10 μM) or ISO (10 μM) for 6 h in the absence or presence of pretreatment (1 h) with H89 (20 μM) or SB (5 μM) as indicated, RNA was recovered from the cells to measure PGC-1α and cyclophilin (Cyclo) transcripts. The level of 18S RNA was visualized by staining with methylene blue in the blot as noted. (B) Effect of PKA and p38 MAPK inhibitors on the activity of the PGC-1α promoter. HIB-1B cells were transfected with PGC-1α-CAT vector as described in Materials and Methods. Cells were treated with FSK (10 μM) for the last 6 h of transfection with or without a preexposure to H89 (20 μM), Rp-cAMP (Rp) (1 mM), or SB (5 μM) for 1 h. The CAT activities were measured and normalized to luciferase. The results shown are means ± standard deviations of two independent experiments, each performed in triplicate. (C) ATF-2 binds to the promoter region of PGC-1α containing CRE. HIB-1B cells were differentiated for 5 days as detailed in Materials and Methods. Cells were then stimulated with FSK for 6 h, followed by incubation with 1% formaldehyde to cross-link DNA and transcriptional factors and histones. DNA fragments sheared by sonication were precipitated with antibodies against ATF-2, CREB, or PPARγ, and the presence of the proximal region of the PGC-1α promoter in the precipitates was detected by PCR with specific primers. Results shown are representative of three independent experiments.

FIG. 7.

Schematic diagram of the signaling pathway of UCP1 transcription in brown fat.