p38β MAPK upregulates atrogin1/MAFbx by specific phosphorylation of C/EBPβ
© Zhang and Li; licensee BioMed Central Ltd. 2012
Received: 9 August 2012
Accepted: 21 September 2012
Published: 9 October 2012
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© Zhang and Li; licensee BioMed Central Ltd. 2012
Received: 9 August 2012
Accepted: 21 September 2012
Published: 9 October 2012
The p38 mitogen-activated protein kinases (MAPK) family plays pivotal roles in skeletal muscle metabolism. Recent evidence revealed that p38α and p38β exert paradoxical effects on muscle protein homeostasis. However, it is unknown why p38β, but not p38α, is capable of mediating muscle catabolism via selective activation of the C/EBPβ that upregulates atrogin1/MAFbx.
Tryptic phosphopeptide mapping was carried out to identify p38α- and p38β-mediated phosphorylation sites in C/EBPβ. Chromosome immunoprecipitation (ChIP) assay was used to evaluate p38α and p38β effect on C/EBPβ binding to the atrogin1/MAFbx promoter. Overexpression or siRNA-mediated gene knockdown of p38α and p38β, and site-directed mutagenesis or knockout of C/EBPβ, were used to analyze the roles of these kinases in muscle catabolism in C2C12 myotubes and mice.
Cellular expression of constitutively active p38α or p38β resulted in phosphorylation of C/EBPβ at multiple serine and threonine residues; however, only p38β phosphorylated Thr-188, which had been known to be critical to the DNA-binding activity of C/EBPβ. Only p38β, but not p38α, activated C/EBPβ-binding to the atrogin1/MAFbx promoter. A C/EBPβ mutant in which Thr-188 was replaced by alanine acted as a dominant-negative inhibitor of atrogin1/MAFbx upregulation induced by either p38β or Lewis lung carcinoma (LLC) cell-conditioned medium (LCM). In addition, knockdown of p38β specifically inhibited C/EBPβ activation and atrogin1/MAFbx upregulation induced by LCM. Finally, expression of active p38β in mouse tibialis anterior specifically induced C/EBPβ phosphorylation at Thr-188, atrogin1/MAFbx upregulation and muscle mass loss, which were blocked in C/EBPβ-null mice.
The α and β isoforms of p38 MAPK are capable of recognizing distinct phosphorylation sites in a substrate. The unique capacity of p38β in mediating muscle catabolism is due to its capability in phosphorylating Thr-188 of C/EBPβ.
The p38 mitogen-activated protein kinases (MAPK) family plays a pivotal role in skeletal muscle by mediating diverse cellular activities, and interestingly, some of which result in paradoxical effects. For example, p38 mediates both insulin stimulation of glucose uptake  and TNF-α stimulation of insulin resistance  in muscle. In the context of skeletal muscle protein homeostasis, p38 responds to both catabolic (lipopolysaccharide, cytokines, and ROS) [3–5] and anabolic (insulin and exercise) stimuli . On one hand, p38 stimulates muscle satellite cell proliferation  and differentiation , which increases muscle mass; on the other hand, p38 stimulates muscle protein degradation leading to muscle atrophy [3–5]. Intriguingly, p38 has the capacity to activate different protein substrates depending on the cellular environment . It is of great interest to understand how the p38 MAPK family is able to mediate the discrete and sometimes opposing effects in response to diverse physiological and pathological stimuli. The family of MAPK is composed of at least four members (α, β, γ and δ), which enable the transduction of a variety of extracellular signals into distinct nuclear responses [9–11]. The α, β, and γ isoforms are found in muscle cells. Recently, it became clear that p38α MAPK plays an essential role in myogenic differentiation [12, 13]. On the other hand, p38γ MAPK appears to regulate the expansion of myogenic precursor cells , endurance exercise-induced mitochondrial biogenesis and angiogenesis , as well as glucose uptake . But, little was known about the function of p38β until our most recent discovery of its role in regulating the atrogin1/MAFbx gene .
Cachexia, a wasting disease characterized by loss of muscle mass with or without loss of fat mass, is frequently associated with such diseases as cancer, sepsis, AIDS, congestive heart failure, diabetes, chronic renal failure and chronic obstructive pulmonary disease (COPD). Cachexia is distinct from starvation-, disuse-, aging-, primary depression-, malabsorption- and hyperthyroidism-induced muscle mass loss and is associated with increased morbidity and mortality [18, 19]. The prominent clinical feature of cachexia is weight loss with anorexia, inflammation, insulin resistance, and increased muscle protein breakdown. Increased muscle protein breakdown in cachexia is at least partially due to accelerated muscle proteolysis by the ubiquitin-proteasome pathway, a common pathway of muscle mass loss due to pathological as well as physiological causes . However, the signaling mechanism of the activation of the ubiquitin-proteasome pathway in cachexia appears to be different from that of physiological muscle atrophy. It is well established that a depression in AKT activity activates FoxO1/3 transcription factors, which upregulates two key ubiquitin ligases (E3 proteins), atrogin1/MAFbx and MuRF1, in animal models of physiological muscle atrophy caused by fasting, denervation and disuse [21–23]. In animal models of cachexia, however, AKT is often activated, which leads to the inactivation of FoxO1/3 [4, 5, 24]. In fact, it has been shown in animal models of cachexia that upregulation of MuRF1 is mediated by NF-κB , and upregulation of atrogin1/MAFbx is mediated by p38 MAPK [4, 5].
We showed most recently that among the known p38 MAPK isoforms only p38β MAPK is capable of upregulating atrogin1/MAFbx via the activation of transcription factor C/EBPβ in response to tumor cell-conditioned medium. In addition, we demonstrated that p38β MAPK upregulation of atrogin1/MAFbx is independent of the AKT-FoxO1/3 signaling pathway . Thus, p38β emerged as a key mediator and a specific therapeutic target of cachexia. Notwithstanding, why p38β is uniquely capable of activating C/EBPβ among the p38 isoforms is unknown. The current study is designed to address the mechanism through which p38β specifically activates C/EBPβ in the context of tumor-induced cachexia. We demonstrate that while p38β shares some common phosphorylation sites with p38α, it specifically phosphorylates the Thr-188 residue of C/EBPβ, which activates C/EBPβ binding to the atrogin1/MAFbx promoter and upregulates this gene in response to a tumor.
Tryptic phosphopeptide mapping was carried out to identify p38α- and p38β-mediated phosphorylation sites in C/EBPβ. ChIP assay was used to evaluate p38α and p38β effect on C/EBPβ-binding to the atrogin1/MAFbx promoter. Overexpression or siRNA-mediated gene knockdown of p38α and p38β, and site-directed mutagenesis or knockout of C/EBPβ, were used to analyze the roles of these kinases in muscle catabolism in C2C12 myotubes and mice.
HEK293T cells (American Type Culture Collection, Manassas, VA, USA) cultured in 150 mm culture plates that were ~50% confluent were co-transfected with a plasmid encoding LAP with a FLAG tag (Addgene) and a plasmid encoding constitutively active p38α or p38β  (10 μg each) using deacylated polyethylenimine (PEI) 22000 , a gift from Dr. Guangwei Du (University of Texas Health Science Center at Houston, Houston, TX, USA). The cell culture medium was replaced with fresh medium at 24 h. Cells were lysed in RIPA buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS, 2 mM phenylmethylsulphonylfluoride (PMSF), 0.5% sodium deoxycholate, 1 mM NaF, 1/100 protease inhibitor cocktail, and 1/100 phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) after an additional incubation of 24 h. LAP in cell lysates was precipitated using FLAG-M2 magnetic beads (Sigma-Aldrich) and subjected to 10% SDS-PAGE. The gel was then stained with Coommassie Blue R-250. The LAP band was cut out and subjected to tryptic phosphopeptide mapping conducted by Taplin Mass Spectrometry Facility at Harvard Medical School using an LTQ-Orbitrap mass spectrometer (Thermo Electron, West Palm Beach, FL, USA).
Murine C2C12 myoblasts (American Type Culture Collection, Manassas, VA, USA) were cultured in growth medium (DMEM supplemented with 10% fetal bovine serum) at 37°C under 5% CO2. At approximately 85 to 90% confluence, myoblast differentiation was induced by incubation for 96 h in differentiation medium (DMEM supplemented with 4% heat-inactivated horse serum) to form myotubes. Plasmids encoding constitutively active p38 isoforms  or a C/EBPβ mutant (p3xFlag-CMV-10-LAP-T188A) were transfected into C2C12 myoblasts of 50% confluence at 1 μg/well in six-well plates using deacylated polyethylenimine (PEI) 22000. At 24 h we induced the cells to differentiate by switching them to differentiation medium. When indicated, myotubes were treated with Lewis lung carcinoma cell (National Cancer Institute, Bethesda, MD, USA)-conditioned medium (LCM, 25% final volume)  or directly harvested. Cell lysate was prepared using the RIPA buffer for further analyses.
Chromosome immunoprecipitation (ChIP) assay was performed as previously described .
A plasmid encoding C/EBPβ in which Thr-188 was replaced by alanine in the pcDNA3 vector (a gift from Dr. Qi-Qun Tang of Fudan University, Shanghai, China) was digested at BamHI and EcoRI restriction sites to release the cDNA insert. That insert was then subcloned into the p3xFlag-CMV-10 vector (Sigma-Aldrich) at the same restriction sites to generate plasmid p3xFlag-CMV-10-LAP-T188A.
Cell and muscle lysate were prepared and western blot analysis was carried out as described previously . Antibodies for total and/or phosphorylated ATF2, FoxO1 (Thr-24)/FoxO3a (Thr-32) and C/EBPβ phosphorylated at Thr-188 were from Cell Signaling Technology (Danvers, MA, USA). Antibody for atrogin1/MAFbx was from ECM Biosciences (Versailles, KY, USA). Antibodies to C/EBPβ (H-7) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to the HA tag was from Covance (Princeton, NJ, USA). Data were normalized to GAPDH.
p38α-specific siRNA (5′-CUCCUUUACUAUCUUUCUCAA-3′) and p38β-specific siRNA (5′-GUCCUGAGGUUCUAGCAAAdTdT-3′) were synthesized by Sigma-Aldrich and transfected into C2C12 myoblasts by electroporation (5 μg/1 × 107 cells) with the Nucleofector system (Lonza, Walkersville, MD, USA), according to the manufacturer’s protocol. Control siRNA was obtained from Ambion (Austin, TX, USA). Differentiation was induced 24 h after transfection.
Experimental protocols were approved in advance by the institutional Animal Welfare Committee at the University of Texas Health Science Center at Houston. C/EBPβ−/− mice in C57BL/6 background were bred from C/EBPβ−/+ mice generated by Dr. Peter Johnson of NCI . While the mice were under anesthesia, plasmids encoding constitutively active p38α or p38β were injected into the tibialis anterior (TA) of the right leg for each mouse (100 μg in 50 μl), and the empty vector pcDNA3.1 was injected into the left leg as control. Immediately after plasmid injection, TA was electroporated by applying square-wave electrical pulses (100 V/cm) eight times with an electrical pulse generator (Model 830, BTX) at a rate of one pulse per second, with each pulse being 20 ms in duration, through a pair of stainless steel needles that were 5 mm apart. The above transfection procedure was repeated in 7 days. In another 7 days, the mice were sacrificed and TAs were collected for analysis.
Data were analyzed with one-way ANOVA or student t test using the SigmaStat software (Systat Software, Point Richmond, CA, USA) as indicated. When applicable, control samples from independent experiments were normalized to a value of 1 without showing variations (actual variations were within a normal range). A P value <0.05 was considered to be statistically significant. Data are presented as the mean ± S.E.
Expression of constitutively active p38 α or p38β resulted in the phosphorylation of diverse amino acid residues in C/EBPβ
Phosphorylated amino acid residues in C/EBPβ
Ser182, Ser183, Ser190, Thr188
Ser64, Ser184, Ser222, Ser276
The selective activation of substrates by various p38 MAPK isoforms was previously attributed to preferential activation of the isoforms by specific MAPK kinase as well as compartmentalization of the isoforms. For example, while p38α is activated by MKK3, MKK6 and MKK4, p38β is activated by MKK6 [10, 35]. However, these may not explain the specific activation of C/EBPβ by p38β, because both p38α and p38β are present in the nucleus and activated by MKK6. The current study demonstrates that the selective activation of substrates by p38 MAPK isoforms is further realized by their recognition of specific phosphorylation sites within a substrate.
Particularly, we show that p38β specifically mediates the phosphorylation of C/EBPβ required for the activation of its binding to the atrogin1/MAFbx promoter. Previous studies indicated that phosphorylation of Thr-188 in C/EBPβ by ERK1/2 MAPK  or cdk2/cyclinA  primes C/EBPβ for subsequent phosphorylation on Ser-184 or Thr-179 by glycogen synthase kinase 3β (GSK3β), which activates the DNA-binding and transactivation functions of C/EBPβ . Our data presented here demonstrate that p38β has the unique capability of mediating dual phosphorylation at both Thr-188 and Ser-184, resulting in the activation of C/EBPβ binding to the atrogin1/MAFbx promoter. In contrast, p38α that mediates phosphorylation of Ser-184 but not Thr-188 is unable to activate C/EBPβ binding to the atrogin1/MAFbx promoter. Because GSK3β was previously shown inactivated by p38 MAPK-mediated phosphorylation , it is unlikely that GSK3β mediates C/EBPβ phosphorylation at Ser-184 in response to p38α and p38β activation.
In the present study we also present the first evidence that overexpression of active p38β in muscle induces muscle catabolism, demonstrating a direct effect of p38β on muscle catabolism in vivo. Previous studies involving systemic activation of p38 in tumor-bearing mice  or septic mice  did not allow such a conclusion.
Although p38β is widely distributed in various tissues  its function is largely unknown, especially when it is compared to p38α, which is not only responsible for the known roles of p38 in inflammatory responses [38, 39] but also in the regulation of myogenesis [12, 13]. The present study demonstrates that activation of p38β, not p38α, induces atrogin1/MAFbx upregulation and muscle mass loss via specific phosphorylation of C/EBPβ, which explains at the molecular level why p38 is capable of playing the seemingly opposing roles in muscle protein homeostasis (promoting myogenesis versus promoting muscle catabolism). Further, these data support p38β as a selective therapeutic target of cachexia. Because C/EBPβ is activated by a number of kinases and regulates a wide variety of genes , it may not be suitable as a drug target. On the other hand, p38β has few known functions, therefore, specific inhibitors of p38β MAPK would be highly desirable for the intervention of cancer cachexia. Unfortunately, only p38α/β-dual or p38α-specific inhibitors are available at the present time.
Because p38β is highly expressed in the heart , it may also regulate the protein homeostasis in heart muscle via influencing C/EBPβ−regulated atrogin1/MAFbx expression. Consistent to this notion, it has been shown recently that exercise induces a reduction in C/EBPβ in cardiomyocytes, which mediates cardiomyocyte hypertrophy . In addition, the transactivation activity of C/EBPβ is suppressed by insulin, an anabolic hormone . Therefore, diverse signaling pathways that regulate protein homeostasis in striated muscles may converge upon C/EBPβ.
The present study demonstrates that the α and β isoforms of p38 MAPK recognize distinct phosphorylation sites in a substrate. p38β MAPK has the unique capacity to mediate the dual phosphorylation of Thr-188 and Ser-184 in C/EBPβ, thereby, activating this transcription factor and inducing muscle catabolism. Therefore, p38β MAPK should be considered as a therapeutic target for cachexia.
Chronic obstructive pulmonary disease
Dulbecco’s modified Eagle’s medium
Lewis lung carcinoma cell-conditioned medium
Lewis lung carcinoma
Mitogen-activated protein kinase
MAPK kinase 6
Polymerase chain reaction
Small interfering RNA
tumor necrosis factor-α.
This study was supported by an R01 grant from National Institute of Arthritis and Musculoskeletal and Skin Diseases to Y-P Li (AR052511). We thank Peter Johnson of National Cancer Institute for sharing the C/EBPβ−/− mice, David Engelberg of Hebrew University for sharing plasmids encoding the constitutively active mutants of p38 MAPK isoforms, and Qi-Qun Tang of Fudan University for sharing plasmid encoding C/EBPβ mutantpcDNA3-LAP-T188A.
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