Among the coordinated cellular events triggered by catecholamines to elicit nonshivering thermogenesis in brown fat, the increased transcription of the UCP1 gene has a clear dependence on β-adrenergic activation of cAMP production (see references 27
for review), but the components of the signaling cascade and the transcription factors beyond this point have been ambiguous. Our findings from this study, together with previous observations (4
), identify p38 MAPK as a central obligatory component of the signaling cascade mediating β-adrenergic regulation of UCP1 expression in BAT and cold acclimation. By a combination of in vivo and in vitro approaches, we showed that p38 MAPK regulates UCP1 gene transcription through a coordinated activation of nuclear factors on two separate elements (PPRE and CRE2) of the UCP1 enhancer region (Fig. ). Specifically, cAMP-regulated transactivation of the UCP1 promoter requires p38 MAPK phosphorylation of both ATF-2 and PGC-1α, while CREB also plays an important role in a PKA-dependent, but p38-independent, manner. Finally, we showed that in addition to its acute activation of PGC-1α by phosphorylation, p38 MAPK also conveys the cAMP signal for increasing the overall expression of PGC-1α to further enhance mitochondrial thermogenic capability (42
Schematic diagram of the signaling pathway of UCP1 transcription in brown fat.
From several different approaches, we demonstrated that the ability of p38 MAPK to control UCP1 gene transcription in brown adipocytes utilizes PGC-1α as a direct target, and this is accomplished at two levels: phosphorylation of the PGC-1α protein as well as an increase in its overall level of expression. Puigserver et al. previously showed that one of the underlying mechanisms for increased skeletal muscle mitochondrial respiration by inflammatory cytokines was phosphorylation of PGC-1α by p38 MAPK (31
). Here we demonstrated that in brown adipocytes, this same phosphorylation of PGC-1α by p38 MAPK is also necessary for driving UCP1 gene transcription, but in this case the stimulus is cAMP and PKA. While the ability of cytokines such as tumor necrosis factor alpha or the interleukins to activate the p38 MAPK signaling cascade is fairly well established (see reference 15
for review), the process whereby PKA ultimately triggers p38 MAPK represents a new pathway that is currently the focus of our efforts. Thus, although the initial stimulus for p38 MAPK activation is different for brown adipocytes and myocytes, the net effect on PGC-1α coactivator function is the same (see Fig. and reference 31
). On the contrary, the transcriptional control of the PGC-1α gene is different among tissues. Expression of the PGC-1α gene in adipocytes was originally shown to be under the control of β-adrenergic stimulation and cAMP (33
), and the transcription factor(s) mediating this response was presumed to be CREB acting downstream of PKA. Instead, our studies show that in brown adipocytes, the induction of PGC-1α by cAMP requires p38 MAPK. Using in vitro and in vivo experiments, we demonstrated that the downstream effector of p38 MAPK in brown adipocytes is ATF-2 rather than CREB. Therefore, the manner by which PGC-1α is regulated in brown adipocytes contrasts with the mechanism in hepatocytes, where CREB phosphorylation is essential (44
We also show that ATF-2 not only regulates the expression of the PGC-1α gene but also is directly involved in the control of UCP1 gene transcription. Using mutagenesis studies and ChIP assays, we showed that both the PPRE and CRE2 elements in the UCP1 enhancer region are required for cAMP- and p38 MAPK-dependent transcription, consistent with earlier studies that showed these two regions were required for the norepinephrine stimulation of the UCP1 promoter (18
). However, contrary to a general assumption that CREB is the vital mediator of β-adrenergic stimulation of UCP1 transcription, our results indicate that ATF-2, but not CREB, is required to regulate the critical enhancer region of the UCP1 promoter (5
). In addition, we show that as an essential player, p38 MAPK regulates and coordinates the activities of the factors bound to both the PPRE and CRE2 by driving the expression and activity of PGC-1α and ATF-2 activation, respectively. One model based upon these results is that the activities of the PPRE and CRE2 are independently sensitive to p38 MAPK, in similarity to a safe-deposit box that requires two separate keys to open. Alternatively, since PGC-1α has been shown to increase transcriptional activity on a model promoter through the assembly of a complex that includes the histone acetyltransferase steroid receptor coactivator 1 and CREB/p300 (30
), we do not exclude the possibility of a more intimate association between PGC-1α and ATF-2 that precisely regulates UCP1 expression, and these aspects are under investigation. Finally, using ChIP studies, we provide evidence that CREB indeed contributes to the cAMP responsiveness of the UCP1 gene by binding the CRE (presumably CRE4) at the proximal region of the promoter. This observation is consistent with a previous report that CREB can interact with the equivalent region of the rat UCP1 promoter (45
Many key features of the brown fat thermogenic response have been shown to be dependent on β-adrenergic stimulation of cAMP production, including proliferation (3
), UCP1 and type II deiodinase expression, mitochondrial biogenesis, and respiration (27
). Whether all these events are regulated by p38 MAPK in the same way as UCP1 and PGC-1α expression in brown fat should now be fully explored.