In previous studies we demonstrated that p38 MAPK activity is involved in the βAR- and cAMP-dependent induction of the Ucp1
gene in brown adipocytes (7
). However, neither the identity of the p38 MAPK nor the molecular intermediaries linking PKA to the activation of p38 MAPK are known. Therefore, we used a series of experiments designed to define the p38 MAPK isoforms and immediate upstream activators involved. In our earlier studies, we presumed that the elevated cAMP levels generated in response to β-agonist stimulation are activating PKA, concluding that this kinase is solely responsible for conveying the cAMP signal that leads to p38 MAPK activation and Ucp1
gene expression. This conclusion was based on the ability of two mechanistically different “inhibitors” of cAMP, the competitive antagonist Rp-cAMPS and the catalytic inhibitor H89, to suppress both 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 activate the small G-protein Rap1 through its interaction with a family of guanine nucleotide exchange factors (GEFs) that include Epac (exchange protein directly activated by cAMP), cAMP-GEF-I, and cAMP-GEF-II (1
). The activities of these molecules are blocked by Rp-cAMPS but are unaffected by H89. Importantly, Rap1 has been shown to be an activator of p38 MAPK (30
). Therefore, as we began this series of studies it was necessary to unequivocally determine whether PKA or a GEF (or some combination of both) leads to stimulation of p38 MAPK and Ucp1
gene expression. We treated HIB-1B brown adipocytes with the β3
AR agonist, CL316,243 (CL), or the adenylyl cyclase stimulator, forskolin (Forsk), in the presence of H89 or Rp-cAMPS or the p38 MAPK inhibitor SB202190 (SB). As shown in Fig. , PKA was activated by either CL or Forsk. The response to both activators was blocked by H89 or Rp-cAMPS but not by SB. Therefore, these results indicate activation of PKA and additionally show that inhibition of p38 MAPK does not affect PKA. As shown in Fig. , neither CL nor Forsk was able to elicit GTP loading to Rap1. Together these results strongly support the conclusion that p38 MAPK activation by cAMP does not depend upon a cAMP-GEF and Rap1 activation but, rather, solely requires PKA.
FIG. 1. β-Adrenergic receptor (βAR) 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 β (more ...)
To confirm the role of both PKA and p38 MAPK in UCP1 induction, HIB-1B cells were pretreated with H89, Rp-cAMPS, or SB, followed by stimulation with either CL or Forsk. Activation of p38 MAPK was measured using glutathione-S-transferase (GST)-tagged ATF-2 as a substrate. As shown in Fig. , p38 MAPK enzyme activity stimulated by CL or Forsk was abrogated by H89, Rp-cAMPS, and SB. These inhibitors similarly blocked the transactivation of the UPC1 enhancer in response to CL and Forsk (Fig. ). These results indicate that, irrespective of the stimulus, PKA is necessary for p38 activation and that, in turn, induction of UCP1 enhancer activity requires p38 MAPK activity.
Since all three βARs are expressed in brown adipocytes and can stimulate cAMP production (56
), we proposed that all of them can activate p38 MAPK and UCP1 transcription. To test this hypothesis, we treated HIB-1B cells with specific agonists of these receptors. As shown in Fig. , the β1
AR-selective agonist, dobutamine, and the β2
AR-selective agonist, salbutamol, both activated p38 MAPK in a PKA-dependent manner. The nonselective βAR activator isoproterenol and the natural adrenergic agonist norepinephrine also activated p38 MAPK in a PKA-dependent fashion (Fig. ). Furthermore, these four βAR agonists also induced UCP1 enhancer activation, which was blocked by p38 MAPK inhibition (Fig. ). These results show that all three βAR subtypes can stimulate p38 MAPK activity and subsequently Ucp1
To determine whether β3
AR stimulation leads to p38 MAPK activation and to a p38 MAPK-dependent Ucp1
gene expression in brown fat in vivo, SB (12.5 mg/kg of body weight) and CL (1 mg/kg) were administered to mice. Phosphorylation and activation of p38 MAPK and JNK was assessed by Western blotting and kinase assays and Ucp1
gene expression by real-time PCR. As shown in Fig. , CL treatment induced phosphorylation of p38 MAPK by 2.5- ± 0.2-fold and p38 MAPK enzyme activity by 2.4- ± 0.1-fold. In contrast, following CL treatment, phosphorylation of JNK could not be detected (Fig. ). The ability of antibody to recognize phospho-JNK was confirmed by treating HIB-1B cells with 5 μg/ml anisomycin for 15 min (Fig. ). Under these same treatment conditions, CL injection stimulated Ucp1
gene expression, and this stimulation was largely prevented (70%) by prior p38 MAPK inhibition (Fig. ). Consistent with what we have previously reported (7
), these results clearly show that selective β3
AR agonist stimulation in vivo triggers p38 MAPK activity to regulation of Ucp1
FIG. 2. β3AR 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 (more ...)
To identify the p38 MAPK isoforms(s) responsible for UCP1 enhancer activation, we first assessed which SB-sensitive isoforms were expressed in BAT and in the brown adipocyte cell line used to dissect the molecular pathway between PKA and Ucp1 gene expression. As shown in Fig. , both p38α and -β mRNAs were expressed in BAT as well as in HIB-1B cells. Consistent with this finding, both proteins were detected by Western blot (Fig. ). We next overexpressed the p38α or -β isoforms in HIB-1B cells and measured UCP1 promoter activity. As shown in Fig. , both isoforms could stimulate UCP1 enhancer activation equally (with a slight preference for the α isoform). Next, we coexpressed MKK6E (a constitutively active form of this kinase that can phosphorylate and activate p38 MAPK) with either p38α or -β in HIB-1B cells, followed by measurements of UCP1 enhancer activity. As shown in Fig. , MKK6E could activate either of the p38 isoforms as measured by significant amplification of UCP1 enhancer activity, but there was a greater preference for p38α MAPK. Together, these results indicate that both p38α and -β isoforms are capable of stimulating UCP1 transcription and that under conditions of maximal stimulation p38α MAPK might couple more efficiently to UCP1 induction. However, these data do not indicate whether either or both isoforms play a role under adrenergic stimulation. To address this issue, we introduced FLAG-tagged p38α or -β MAPK into HIB-1B cells and subsequently treated the cells with CL or Forsk. As clearly shown in Fig. , p38α MAPK but not p38β was activated by CL or Forsk. We also performed immunoprecipitation experiments of the endogenous p38 MAPK isoforms and confirmed that Forsk-induced p38 MAPK activity could be recovered only from the p38α MAPK immunoprecipitate (Fig. ). Interestingly, using our brown adipocyte model, transactivation of the UCP1 enhancer by CL or Forsk was potentiated only by p38α but not by the p38β isoform (Fig. ). In order to validate this selectivity in vivo, mice were either injected with 1 mg/kg CL or exposed to a 4°C environment. As shown in Fig. , both manipulations led to the sole activation of the p38α MAPK isoform in interscapular BAT. Altogether, these data establish that p38α MAPK but not p38β is activated during sympathetic nervous system stimulation of the thermogenic program and following exposure of BAT and brown adipocytes to sympathomimetic drugs.
FIG. 3. p38α or p38β MAPK can promote UCP1 enhancer activity. The expression of p38α and p38β MAPKs was measured in BAT and HIB-1B cells by quantitative real-time PCR (A) and Western blotting with selective antibodies for each (more ...)
FIG. 4. p38α MAPK is selectively activated following β-adrenergic or forskolin stimulation. (A and B) HIB-1B cells were transfected with the β3AR and with FLAG-tagged p38α MAPK (A) or FLAG-tagged p38β MAPK (B). (A and B) (more ...)
Establishing that the α isoform of p38 MAPK is the one that is activated, we used siRNA gene silencing to demonstrate that p38α MAPK and not p38β MAPK was responsible for the induction of UCP1 expression. In these studies it was first necessary to demonstrate the efficacy of the siRNAs directed against either the p38α or -β isoforms. This was examined in HIB-1B cells. As shown in Fig. , the siRNA against either form of p38 MAPK reduced the targeted protein level by more than 80% without affecting the other isoform. More importantly, as shown in Fig. , using this approach we found that essentially all CL- and Forsk-promoted p38 activity can be attributed to the p38α MAPK isoform. Figure further shows that the siRNA against p38α MAPK completely inhibited UCP1 enhancer activation, while the siRNA against p38β failed to do so. Similar results were obtained in experiments employing dominant-negative constructs of p38α and p38β MAPK (not shown). Finally, it was rather remarkable to find that even the 60- to 80-fold induction of the endogenous Ucp1 gene in HIB-1B cells was totally eliminated by the p38α MAPK siRNA (Fig. ). Altogether these data leave little doubt about the highly specific activation of p38α MAPK by βAR agonists and PKA and its essential role in the activation of the Ucp1 gene.
FIG. 5. p38α MAPK is essential for β3AR agonist or Forsk induction of Ucp1 gene expression. (A and B) HIB-1B cells were transfected with siRNA targeting p38α and p38β MAPK, and 48 h later kinases were immunoprecipitated with specific (more ...)
The next objective was to address the origin of p38α MAPK selectivity. For this purpose we explored which of the MKKs were activated by CL and Forsk. In Fig. (middle panel), MKK3 and/or MKK6 was phosphorylated in a PKA-dependent manner following CL or Forsk. However, neither CL nor Forsk was able to promote the phosphorylation of MKK4 or MKK7 (Fig. ). These results are consistent with the fact that the JNK pathway is not activated in brown adipose tissue in vivo (Fig. ) or in brown adipocytes in vitro (Fig. ). However, the nonisoform selective nature of the MKK3/6 phospho-antibody required additional experimentation in order to address whether one (or both) of these two MKK isoforms was activated. We performed selective immunoprecipitation of MKK3 and MKK6 under basal and cAMP-stimulated conditions. As shown in Fig. , Forsk-induced phospho-MKK3/6 immunoreactivity was detected only upon immunoprecipitation of MKK3. Confirmation of this exclusive activation of MKK3 versus MKK6 in vivo was obtained from BAT samples of mice treated with CL or placed in a 4°C environment. Collectively, these findings demonstrate a selective activation at the level of the direct upstream kinase within the p38 MAPK module, which might be an underlying mechanism of the above-described specific p38α MAPK activation.
FIG. 6. β-Adrenergic agonists and cAMP promotes activation of MKK3. (A and B) HIB-1B cells were treated as described in Fig. and then lysed for measurement of the activity and amounts of the indicated kinases by Western blotting as detailed (more ...)
MKK6 is a universal p38 MAPK activator, but the ability of MKK3 to activate p38β MAPK is modest at best (18
). It was tempting to speculate that the specific involvement of p38α MAPK would be recapitulated at the level of MKK3. We therefore tested the functional impact of overexpressing MKK3, MKK4, MKK6, and MKK7 individually on UCP1 enhancer activity and p38 MAPK activity. The results clearly show that MKK3, and to a much lower extent MKK6, can potentiate the effects of CL and Forsk on both UCP1 expression (Fig. ) and p38 MAPK activity (Fig. ). The reciprocal experiments, using siRNAs that specifically downregulated MKK3 and MKK6 by more than 80% (Fig. ), show that only the selective siRNA directed against MKK3 could block CL and Forsk induction of p38 MAPK activity (Fig. ) as well as UCP1 expression (Fig. ). Finally, the siRNA that specifically targets MKK3 was the only one capable of completely interfering with the expression of the endogenous Ucp1
gene (Fig. ). All together, these results unequivocally define the proximal steps in the cAMP- and PKA-dependent activation of the Ucp1
gene in brown fat as being mediated solely by MKK3 and p38α MAPK.
FIG. 7. MKK3 is essential for β3AR agonist or Forsk induction of Ucp1 gene expression. (A) HIB-1B cells were transfected with the β3AR, with FLAG-tagged MKK3, MKK4, MKK6, or MKK7, and with the enhancer-CAT plasmid. Two days later the cells were (more ...)
The MAP kinases are usually assembled together with their upstream MKKs into a signaling module that is coordinated by large scaffolding molecules such as the JIPs (JNK-interacting proteins) (21
). As a result, these scaffold proteins can concentrate interacting signaling partners in the vicinity of an upstream activator in order to favor a particular pathway. Since there is no established link between PKA and p38 MAPK, we attempted to determine which, if any, of the known JIP family members in brown adipocytes might serve as the scaffold to specifically bind p38α MAPK and MKK3. The first objective was to determine the relative expression levels of the four known JIPs: JIP1, JIP2, JIP3, and JLP. As shown in Fig. , there was no detectable expression of JIP1 in 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. ). Based on these results, each of these three JIPs was analyzed for its ability to interact specifically with MKK3 and p38α MAPK. His-tagged or S-protein-tagged constructs of JIP2, JIP3, and JLP were transfected into HIB-1B cells, followed by their immunoprecipitation in order to assess the identity of any interacting kinases. Figure clearly shows that only JIP2 was able to specifically recover both MKK3 and p38α MAPK. It is noteworthy that neither MKK6 nor p38β MAPK was found under any circumstance, although all these molecules are clearly present as shown in the lysate.
FIG. 8. p38α 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 (more ...)