The FASEB Journal • Research Communication
Signaling mechanism of tumor cell-induced up-regulation of E3 ubiquitin ligase UBR2 Guohua Zhang,* Ren-Kuo Lin,* Yong Tae Kwon,†,‡,§ and Yi-Ping Li*,1 *Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas, USA; †Center for Pharmacogenetics and ‡Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; and §World Class University Program, Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology and College of Medicine, Seoul National University, Seoul, Republic of Korea The N-end rule pathway contributes significantly to accelerated muscle proteolysis mediated by the ubiquitin-proteasome pathway in various catabolic conditions. UBR2 (aka E3␣-II) is the only known E3 ubiquitin ligase of the N-end rule pathway that is up-regulated by cachectic stimuli including proinflammatory cytokines and tumors. However, the signaling mechanism through which UBR2 is up-regulated remains undetermined. Here we identify a signaling pathway that mediates tumor cell-induced up-regulation of UBR2. UBR2 expression in C2C12 myotubes was up-regulated by conditioned medium from Lewis lung carcinoma cells or C26 colon adenocarcinoma cells, which was blocked by a pharmacological inhibitor of p38␣/ mitogen-activated protein kinase (MAPK), SB202190. Similarly, SB202190 administration (i.p.) abolished UBR2 up-regulation in the tibialis anterior of LLC tumor-bearing mice. Genetic gain and loss of function assays in C2C12 myotubes indicated that tumor-induced activation of the p38 isoform is sufficient and necessary for UBR2 up-regulation. In addition, UBR2 up-regulation required p38-mediated phosphorylation of CCAAT/enhancer binding protein (C/EBP)- Thr-188, which was critical to C/EBP binding to the UBR2 promoter. Furthermore, luciferase reporter assay revealed that the C/EBP binding motif in the UBR2 promoter is a functional C/EBP-responsive cis-element that enhances the promoter activity on activation by p38. Finally, genetic ablation of C/EBP blocked UBR2 up-regulation in LLC tumor-bearing mice. These results suggest that UBR2 up-regulation in cachectic muscle is mediated by the p38-C/EBP signaling pathway responsible for the bulk of tumorinduced muscle proteolysis.—Zhang, G., Lin, R.-K., Kwon, Y. T., Li, Y.-P. Signaling mechanism of tumor
ABSTRACT
Abbreviations: CCM, C26 adenocarcinoma cell-conditioned medium; C/EBP, CCAAT/enhancer binding protein; ChIP, chromatin immunoprecipitation; IL-6, interleukin 6; LCM, Lewis lung carcinoma cell-conditioned medium; LLC, Lewis lung carcinoma; MAPK, mitogen-activated protein kinase; MKK6, mitogen-activated protein kinase kinase 6; siRNA, small interfering RNA; TA, tibialis anterior; TNF-␣, tumor necrosis factor ␣; UPP, ubiquitin-proteasome pathway 0892-6638/13/0027-2893 © FASEB
cell-induced up-regulation of E3 ubiquitin ligase UBR2. FASEB J. 27, 2893–2901 (2013). www.fasebj.org Key Words: cachexia 䡠 p38 䡠 MAPK 䡠 C/EBP 䡠 gene regulation The ubiquitin-proteasome pathway (UPP) plays a major role in the muscle wasting associated with cachexia, including cancer-induced cachexia (1, 2). The up-regulation of specific E3 ubiquitin ligases that attach ubiquitin to targeted substrates is a rate-limiting step for protein degradation by the UPP in muscle wasting (3). Thus, muscle-specific E3 ubiquitin ligases that are up-regulated in cancer hosts are potential therapeutic targets of muscle wasting. However, such E3 ubiquitin ligases are often expressed constitutively and may have physiological roles in the maintenance of skeletal muscle (4). Therefore, blocking their pathological upregulation would be a more feasible therapeutic strategy than direct inhibition of the enzymes per se. To date, atrogin1/MAFbx and MuRF1 are the two best-studied E3 ubiquitin ligases implicated in muscle catabolism induced by various physiological and pathological stimuli. However, they do not account for all the muscle mass loss in animal models of muscle atrophy (3). While MuRF1 directly targets such myofibrillar proteins as myosin and ␣-actin for degradation (5, 6), atrogin1/ MAFbx indirectly reduces muscle mass by targeting such regulatory proteins as eukaryotic initiation factor 3 subunit 5 (eIF3-f; refs. 7, 8) and MyoD (9), resulting in lower expression of such myofibrillar protein as myosin (10). Notably, atrogin1/MAFbx and MuRF1 are not always up-regulated in parallel. In some cachectic cancer models (10, 11) as well as some human catabolic conditions (12, 13) where myofibrillar protein loss takes place only atrogin1/MAFbx, but not MuRF1, is up-regulated. The up-regulation of atrogin1/MAFbx alone could not explain the profound increase in muscle protein degradation observed in tumor-bearing 1 Correspondence: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX 77030, USA. E-mail:
[email protected] doi: 10.1096/fj.12-222711
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mice, which suggests the involvement of additional E3 ligases that target myofibrillar proteins in cancer cachexia. In physiological muscle atrophy induced by starvation, denervation, or disuse, atrogin1/MAFbx and MuRF1 up-regulation is mediated by FoxO1/3 transcription factors, which are negatively regulated by the PI3K/AKT signaling pathway (14 –16). However, cachectic tumor cells, such as C26 colon adenocarcinoma (17) and Lewis lung carcinoma (LLC; ref. 10), induce AKT activation resulting in FoxO1/3 inactivation in muscle cells. LLC cell-induced atrogin1/MAFbx upregulation is independent of the 2 FoxO1/3-responsive cis-elements in its promoter; rather, it is dependent on a p38 mitogen-activated protein kinase (MAPK)-activated CCAAT/enhancer binding protein (C/EBP)responsive cis-element (10). In addition, p38 activates C/EBP but not FoxO1/3 (18). Furthermore, blocking p38 or C/EBP activation effectively blocks muscle proteolysis in LLC tumor-bearing mice, despite the fact that MuRF1 remains unchanged (10). These data suggest that the p38 MAPK-C/EBP signaling pathway has a central role in mediating muscle catabolism induced by tumor cells, and that this signaling pathway mediates muscle catabolism via regulating unidentified E3 ubiquitin ligases in addition to atrogin1/MAFbx. Protein degradation via the N-end rule pathway accounts for a large portion of total protein ubiquitylation induced by such inflammatory conditions as sepsis, cancer, and diabetes (19 –21). The muscle-expressed functional and structural homologs UBR1 (also known as E3␣; ref. 22) and UBR2 (also known as E3␣-II; refs. 23, 24) are E3 ubiquitin ligases that serve as the substrate recognition components of the N-end rule pathway. UBR1 mediates physiological muscle atrophy induced by starvation or denervation (25); however, it appears to be nonessential to the pathogenesis of cachectic muscle wasting (24). UBR2, on the other hand, is the only known N-end rule pathway E3 that is up-regulated by cachectic stimuli, including tumor and proinflammatory cytokines [tumor necrosis factor ␣ (TNF-␣) and interleukin 6 (IL-6)] and is highly efficient for protein ubiquitylation via the N-end rule pathway (24). Thus, UBR2 may have a particularly important role in mediating the degradation of muscle proteins in cancer cachexia. The present study tested the hypothesis that UBR2 up-regulation by tumor is mediated by the p38 MAPK-C/EBP signaling pathway that is critical to tumor-induced muscle mass loss. Here we present evidence that supports our hypotheses.
MATERIALS AND METHODS
under 5% CO2. At 85–90% 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 (26), wild-type, or a C/EBP mutant (p3xFlag-CMV-10-LAP-T188A) were transfected into C2C12 myoblasts as described previously (18). In 24 h, the cells were induced to differentiate by switching to differentiation medium. Ad5 cytomegalovirus encoding MKK6bE (27), a constitutively active form of MAPK kinase 6 (MKK6), or GFP were transduced into C2C12 myotubes as described previously (28). When indicated, myotubes were treated with 25% final volume of LLC cell [U.S. National Cancer Institute (NCI), Bethesda, MD, USA]conditioned medium (LCM) or C26 adenocarcinoma cell (American Type Culture Collection)-conditioned medium (CCM). Conditioned medium of the nontumorigenic human lung epithelial cell line NL20 (American Type Culture Collection) was used as control. Cell lysate was prepared using the RIPA buffer for further analyses. Animal use Experimental protocols were approved in advance by the institutional Animal Welfare Committee at the University of Texas Health Science Center at Houston. For LLC-induced cancer cachexia model, 100 l LLC cells (5⫻106), or an equal volume of PBS (control) was injected subcutaneously into the right flanks of 8-wk-old male mice (C57BL/6). Mice with tumor size between 1 and 1.5 cm were euthanized in 14 d, and the tibialis anterior (TA) was collected for analysis. When indicated, SB202190 (5 mg/kg) or an equal volume of vehicle (PBS containing 50% DMSO) was injected intraperitoneally (i.p.) daily from d 5 after LLC implant when the tumor became palpable. C/EBP⫺/⫺ mice in C57BL/6 background were bred from C/EBP⫺/⫹ mice generated by Dr. Peter Johnson (NCI; ref. 29). Real-time PCR Real-time PCR was performed as described previously (28) using primers specific for the UBR2 gene (sense: 5=-TATTCTCCTCCTTACCTTG-3=, antisense: 5=-CGAAACCGCTCTTGGCATA-3=). Western blot analysis Cell and muscle lysates were prepared and Western blot analysis was carried out as described previously (10). Antibody to UBR2 was from Novus Biologicals (Littleton, CO, USA). Antibodies to p38␣, p38, MKK6, total and phosphorylated p38 or ATF2, and C/EBP 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/EBP (H-7) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to the HA tag was from Covance (Princeton, NJ, USA). Antibody to FLAG tag was obtained from Sigma-Aldrich (St. Louis, MO, USA). Data were normalized to GAPDH (antibody was from Millipore, Billerica, MA, USA).
Myogenic cell culture, transfection, and transduction
Small interfering RNA (siRNA)-mediated mRNA knockdown
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
C/EBP-specific mouse siRNA (10) and p38␣- and p38specific mouse siRNA (18) were synthesized by SigmaAldrich and transfected into C2C12 myoblasts by electro-
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poration (5 g/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. Chromatin immunoprecipitation (ChIP) assay Examination of the UBR2 gene promoter sequence using the Computational Biology and Informatics Laboratory database (University of Pennsylvania, Philadelphia, PA, USA; http:// www.cbil.upenn.edu/cgi-bin/tess) revealed a conserved putative C/EBP binding motif (TTTTCCAG) in the 5= promoter region of the gene ~150 to 250 bp upstream of the transcription initiation site. To evaluate C/EBP binding to this motif, a ChIP assay was performed as described previously (10) using primers specific for mouse UBR2 promoter region containing the C/EBP-binding motif (forward: 5=-GTTCTT CTGGTGTCAAGCCC-3=, reverse: 5=-CAGGGCATTCTGGGTACTGT-3=), and the flanking region as control (forward: 5=-CTGTGTGTCTATCGCGCCAGGA-3=, reverse: 5=-CGTGAGAGGGGCTTGACACCA-3=). Promoter assays Luciferase reporter gene constructs under the control of the 5=-flanking promoter sequence of the mouse UBR2 gene containing a putative C/EBP-binding motif 5=-TTTTCCAG-3= were constructed by inserting DNA fragment generated by PCR using mouse genomic DNA as the template into the pGL4.10 vector (Promega, Madison, WI, USA). A construct with mutations within the C/EBP-binding motif to 5=-TCTACACG-3= was created by using a site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Primers used for generating these constructs were as follows: pC sense: 5=CAAGAATAAATAGGACTAAG-3=, antisense: 5=-CTTCTCTCCTCCTCCTCTCC-3=; pD sense: 5=-TGGACTACAACTCCCATGAT-3=, antisense: 5=-CTTCTCTCCTCCTCCTCTCC-3=; pC-M sense: 5=-GATTCTAAATTGTCTTTCTACACGCTTCCGCCAGG3=, antisense: 5=- CCTGGCGGAAGCGTGTAGAAAGACAATTTAGAATC-3=. Plasmids were transfected into C2C12 myoblasts as described previously (10). We used plasmid encoding the LAP form of C/EBPb (provided by Peter Johnson, NCI, via Addgene, Cambridge, MA, USA), MKK6bE (a gift from Jiahuai Han, Scripps Research Institute, La Jolla, CA, USA), or isoforms of p38 MAPK (gifts from David Engelberg, Hebrew University, Jerusalem, Israel) along with 0.2 mg of a plasmid encoding Renilla luciferase (phRL-TK-luc, Promega). After culturing in growth medium for 24 h, luciferase activity in cell lysate was measured using the DualLuciferase Reporter Assay system (Promega) in a Synergy 2 Multi-Mode Microplate Reader (Biotek Instruments, Winnoski, VT, USA).
RESULTS Tumor cells induce UBR2 up-regulation via the activation of p38 MAPK Previously, UBR2 was shown to be up-regulated in the muscle of YAH-130 tumor-bearing rats and C26 tumorbearing mice (24). To assess whether tumor cells can directly induce UBR2 up-regulation in muscle cells via the activation of p38 MAPK, we treated C2C12 myotubes with LCM or CCM. We observed up-regulation of the UBR2 mRNA by LCM or CCM in 4 h, and the up-regulation was abolished by pretreatment of myotubes with the p38␣/ MAPK inhibitor SB202190 (Fig. 1A). Similar elevation in UBR2 protein was observed at 8 h of LCM or CCM treatment, which was also blocked by SB202190 (Fig. 1B). To evaluate whether these effects took place in vivo, we treated LLC tumor-bearing mice with SB202190, which we previously showed blocks muscle mass loss without affecting tumor volume (10). UBR2 protein levels increased in TA of LLC tumorbearing mice, and SB202190 prevented the increase (Fig. 1C). These results suggest that LLC or C26 cell-released substance induces UBR2 up-regulation in muscle cells via the activation of ␣ or  isoform of p38 MAPK. To determine which isoform of p38 MAPK mediates tumor cell-induced UBR2 up-regulation, a plasmid encoding constitutively active ␣ or  isoform of p38 MAPK was transfected into C2C12 myoblasts. Although active p38␣ accelerated myoblast differentiation at the early stage of differentiation, at the end of differentiation (96 h) there was no visible difference in the myotubes formed due to the overexpression of active p38 isoforms. Expression of the HA tag and activation of p38 substrate ATF2 were monitored for verification of the expression of active isoforms (Fig. 2A). In myotubes overexpressing active p38, UBR2 protein level increased. However, in myotubes overexpressing active p38␣, UBR2 protein level remained unchanged (Fig. 2A). Conversely, we transfected C2C12 myoblasts with siRNA specific for p38␣ or p38 to knock down the mRNAs individually. No visible difference in the myotubes formed at 96 h was seen due to the knockdowns, despite initial slowdown of the differentiation in p38␣-knockdown myoblasts. The myotubes were subsequently treated with LCM. We found that only p38 knockdown, but not p38␣ knockdown, blocked UBR2 up-regulation by LCM (Fig. 2B). These data suggest that p38 activation by LLC cells is sufficient and necessary for UBR2 up-regulation.
Statistical analysis Data were analyzed with 1-way ANOVA or Student’s t test using 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 value of P ⬍ 0.05 was considered to be statistically significant. Data are presented as means ⫾ se. MECHANISM OF UBR2 UP-REGULATION BY TUMOR
p38 MAPK mediates tumor stimulation of the UBR2 promoter activity via activating C/EBP binding to a cis-element in the promoter Next, we asked how p38 mediates tumor cell-induced UBR2 up-regulation. Because C/EBP is a known p38 substrate in vitro (30) and in C2C12 myotubes (10), we examined the sequence of the UBR2 gene promoter 2895
Figure 1. Tumor cells induce UBR2 up-regulation in a p38 MAPK-dependent manner. A) Tumor cell-conditioned media up-regulate the UBR2 mRNA via p38 MAPK. C2C12 myotubes were pretreated with SB212190 (10 M) or vehicle for 15 min prior to treatment with LCM or CCM for 4 h. UBR2 mRNA levels in the myotubes were determined by real-time PCR. Data were analyzed with ANOVA. *P ⬍ 0.05 vs. vehicle-treated control; † P ⬍ 0.05 vs. vehicle plus tumor cell-conditioned medium. B) Tumor cell-conditioned media up-regulate UBR2 protein via p38 MAPK. C2C12 myotubes were pretreated with SB212190 (10 M) or vehicle for 15 min prior to treatment with LCM or CCM for 8 h. UBR2 protein levels were analyzed by Western blot analysis. Data were analyzed with ANOVA. *P ⬍ 0.05 vs. vehicletreated control; †P ⬍ 0.05 vs. vehicle plus tumor cell-conditioned medium. C) UBR2 is up-regulated in the muscle of LLC tumorbearing mice in a p38 MAPK-dependent manner. LLC cells or PBS (control) were subcutaneously injected into the right flank of adult male C57BL/6 mice. From d 5 of LLC cell implant when tumor became palpable, SB202190 (5 mg/kg) was administered daily (i.p.). On d 14, UBR2 levels in TA were determined by Western blotting Data were analyzed with ANOVA. *P ⬍ 0.05 vs. vehicle-treated control; †P ⬍ 0.05 vs. vehicle injection plus LLC xenograft.
using the Computational Biology and Informatics Laboratory database (http://www.cbil.upenn.edu/cgi-bin/ tess). The search revealed a conserved putative C/EBP binding motif (TTTTCCAG) in the 5= promoter region of the gene ⬃150 to 250 bp upstream of the transcription initiation site (Fig. 3A). On the other hand, unlike the atrogin1/MAFbx promoter that contains 2 FoxObinding motives (10, 14), no putative FoxO-binding motif was found in the 2000 bp of 5= flanking sequence of the UBR2 gene searched. ChIP assay was conducted
to evaluate whether p38 regulates C/EBP binding to the UBR2 promoter. We first examined whether p38 activation stimulates C/EBP binding to the UBR2 promoter by transducing C2C12 myotubes with an adenovirus encoding constitutively active MKK6bE that activates all isoforms of p38 (27). Overexpressed MKK6bE, which activated p38 and phosphorylated C/EBP at Thr-188, indeed activated C/EBP binding to a fragment of the UBR2 promoter containing the putative C/EBP binding motif, while overexpressed
Figure 2. p38 MAPK specifically mediates tumor cell-induced UBR2 up-regulation. A) Activation of p38 is sufficient to up-regulate UBR2. A plasmid encoding HA-tagged active p38␣, p38, or empty vector was transfected into C2C12 myoblasts. After differentiation for 96 h, p38 activation and UBR2 levels were evaluated by Western blotting. Data were analyzed with ANOVA. *P ⬍ 0.05 vs. vector control. B) p38 is necessary for tumor cell-induced up-regulation of UBR2. C2C12 myoblasts were transfected with siRNA of p38␣, p38, or scrambled sequence as control. After differentiation for 96 h, myotubes were treated with LCM for 8 h. p38 expression and UBR2 levels were evaluated by Western blotting. Data were analyzed with ANOVA. *P ⬍ 0.05 vs. control siRNAtransfected cells treated with control medium; † P ⬍ 0.05 vs. p38␣ siRNA-transfected cells treated with LCM.
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Figure 3. Tumor cells induce C/EBP binding to the UBR2 promoter via p38 MAPK in muscle cells. A) The 5= promoter region of the UBR2 gene contains a conserved putative C/EBP-binding motif. B) Activation of p38 MAPK results in C/EBP binding to the UBR2 promoter. Adenovirus encoding MKK6bE or GFP was transduced into C2C12 cells that have been incubated in differentiation medium for 48 h. In an additional 48 h, when myotubes formed, MKK6 expression and activation of p38 and C/EBP were evaluated by Western blotting (top panel). ChIP assay was conducted to evaluate C/EBP binding to the UBR2 promoter (bottom panel). C) Activation of p38 is sufficient to activate C/EBP binding to the UBR2 promoter. A plasmid encoding constitutively active p38␣ or p38 was transfected into C2C12 myoblasts. After differentiation, C/EBP binding to the UBR2 promoter in myotubes was evaluated by ChIP assy. D) p38 MAPK is required for tumor cell-induced C/EBP binding to the UBR2 promoter. C2C12 myotubes formed from differentiated myoblasts that had been transfected with siRNA of p38␣, p38, or scrambled control were treated with LCM for 1 h. ChIP assay was conducted to evaluate C/EBP binding to the UBR2 promoter. E) C/EBP phosphorylation at Thr-188 is crucial for tumor cell-induced C/EBP binding to the UBR2 promoter. Empty vector or a plasmid encoding either wild-type LAP or the LAP-T188A mutant was transfected into C2C12 myotubes. After differentiation, myotubes were treated with LCM or control medium for 1 h. ChIP assay was conducted to evaluate C/EBP binding to the UBR2 promoter.
GFP did not do so (Fig. 3B). We then examined the binding activity in myotubes overexpressing active p38␣ or p38. Only the expression of active p38 resulted in increased C/EBP binding to the UBR2 promoter region containing the putative C/EBP binding motif, but not to the flanking region in the promoter amplified by control primers (Fig. 3C). We further examined the binding activity in myotubes in which p38␣ or p38 was knocked down by siRNA. Deficiency in p38 expression specifically blocked LCM stimulation of C/EBP binding to the UBR2 promoter (Fig. 3D). We demonstrated previously that p38 activates C/EBP binding to DNA by specific phosphorylation of its Thr-188 residue (18). To determine whether p38-mediated phosphorylation of C/EBP is required for UBR2 up-regulation, we transfected C2C12 cells with a plasmid encoding wildtype or a mutant of C/EBP in which Thr-188 was replaced by alanine (LAP-T188A; ref. 18). This mutant blocked LCM-induced C/EBP binding to the UBR2 promoter (Fig. 3E). These results indicate that p38 MAPK mediates tumor cell-induced activation of C/EBP binding to the UBR2 promoter. To further investigate whether the C/EBP binding motif identified in the UBR2 promoter is a functional cis-element that enhances transactivation in response to p38 MAPK activation of C/EBP, we conducted a luciferase reporter assay. As shown in Fig. 4A, a luciferase reporter gene construct under the control of the 5=-flanking promoter sequence of the mouse UBR2 gene was generated (pC) along with constructs in which the C/EBP binding motif TTTCCAG was deleted (pD) or mutated (pC-M). The constructs were MECHANISM OF UBR2 UP-REGULATION BY TUMOR
transfected into C2C12 myoblasts and in 24 h a basal promoter activity was detected. Taking advantage of the fact that C/EBP is not expressed before the differentiation of C2C12 myoblasts (31), the plasmid encoding constitutively active p38 was cotransfected with or without the cotransfection of a plasmid encoding C/EBP (LAP) to determine whether p38 stimulation of the promoter activity requires LAP. As shown in Fig. 4B, active p38 stimulated the promoter activity by 2.8-fold in a LAP-dependent manner, which was abolished by SB202190. However, when the C/EBP binding motif is deleted or mutated, active p38 failed to stimulate the promoter activity. Therefore, the C/EBP binding motif identified in the UBR2 promoter is a bona fide p38-activated C/EBP-responsive cis-enhancer. p38 MAPK-mediated activation of C/EBP is crucial for tumor cell-induced UBR2 up-regulation We further asked whether C/EBP activation by p38 is crucial for tumor cell-induced up-regulation of endogenous UBR2. Utilizing siRNA specific for C/EBP, we knocked down C/EBP in C2C12 myotubes and found that LCM up-regulation of UBR2 was prevented in C/EBP-deficient myotubes (Fig. 5). This result suggests that C/EBP is essential for tumor cell-induced up-regulation of UBR2. To determine whether p38-mediated phosphorylation of C/EBP is required for UBR2 up-regulation, we cotransfected C2C12 cells with active p38␣ or p38 along with LAP-T188A. We observed that LAP-T188A acted as a dominant-negative inhibitor of p38-medi2897
Figure 4. A C/EBP-responsive cis-element in the UBR2 promoter mediates p38 MAPK stimulation of the promoter activity. A) URB2 promoter-directed luciferase reporter plasmids were constructed as illustrated. B) C2C12 myoblasts were transfected with one of the URB2 promoter-directed luciferase reporter plasmids illustrated in panel A, and cotransfected with a plasmid encoding LAP, active p38 or both as indicated. Transfected myoblasts were incubated for 24 h with or without the presence of SB212190 (10 M) before reporter activity was measured. Data were analyzed with ANOVA. *P ⬍ 0.05 vs. control.
ated up-regulation of UBR2 (Fig. 6A). In addition, the expression of LAP-T188A in C2C12 myotubes blocked LCM-induced up-regulation of UBR2, while the expression of wild-type LAP did not do so (Fig. 6B). This result indicates that C/EBP activation by p38 is critical to tumor cell-induced up-regulation of UBR2. We observed previously that LLC tumor-induced weight loss and muscle proteolysis were blocked in C/EBP⫺/⫺ mice, as was atrogin1/MAFbx up-regulation (10). To determine whether tumor-induced UBR2 up-regulation is dependent on C/EBP in vivo, we compared UBR2 levels in the TA of LLC tumor-bearing C/EBP⫺/⫺ mice with that in wildtype littermates. We observed that LLC tumor induced UBR2 up-regulation in wild-type mice but failed to do so in C/EBP-null mice (Fig. 7). This in vivo result corroborates the above in vitro data and allows us to conclude that C/EBP mediates tumorinduced UBR2 up-regulation in muscle.
DISCUSSION The present study reveals for the first time that UBR2, a tumor-induced N-end rule pathway E3 ligase, is 2898
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up-regulated by the p38 MAPK-C/EBP signaling pathway. Since the discovery of atrogin1/MAFbx and MuRF1 and their role in muscle atrophy of rodents over a decade ago, the two E3 ligases have taken the center stage in the perceived mechanism of various forms of muscle atrophy. However, human studies found no elevation of MuRF1 (12, 13) or both MuRF1 and atrogin1/MAFbx (32) in cachectic muscle of diverse catabolic diseases. As shown previously, the p38 MAPK-C/EBP signaling pathway mediates ⱖ75% of LLC tumor-induced muscle proteolysis without affecting MuRF1 expression, which could not be explained by the observed elevation of atrogin1/MAFbx alone that does not directly target muscle proteins for degradation (10). Our data presented here demonstrate that the p38 MAPK-C/EBP signaling pathway mediates tumor cell-induced up-regulation of E3 ligase UBR2, which has a major role in mediating the ubiquitylation of muscle proteins in tumor-bearing mice (24). Based on our current and previous data (10, 18), we propose a working model for the signaling mechanism of tumor cell-induced muscle mass loss, as illustrated in Fig. 8. Although the present study was focused on LLCinduced muscle wasting, we demonstrated that C26 adenocarcinoma cells also induce UBR2 up-regulation via p38 MAPK. In a separate study, we observed that pancreatic cancer cells stimulate muscle protein loss via the activation of p38 MAPK (unpublished results). Therefore, the p38 MAPK-C/EBP signaling pathway responds to multiple types of cachexia-inducing tumors and is potentially important for cachexia induced by multiple types of tumor. In a previous study, we demonstrated that the activation of p38 MAPK is sufficient and necessary for LLC cell-induced muscle mass loss (18). A wide range of stressful stimuli can activate p38 MAPK, including endotoxin (28), cytokines (33), ROS, UV light, irradi-
Figure 5. C/EBP is essential for tumor cell-induced upregulation of UBR2. C2C12 myoblasts were transfected with C/EBP-specific or control siRNA. After 96 h of differentiation, myotubes were treated with LCM for 8 h. Western blotting was conducted to evaluate the levels of C/EBP and UBR2 in the lysate of myotubes. Data were analyzed with ANOVA; *P ⬍ 0.05 vs. control siRNA-transfected cells treated with control medium; †P ⬍ 0.05 vs. control siRNA-transfected cells treated with LCM.
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Figure 6. C/EBP phosphorylation at Thr-188 is critical to UBR2 up-regulation by p38 MAPK and tumor cells. A) C/EBP phosphorylation at Thr-188 is critical to UBR2 up-regulation by p38 MAPK. C2C12 myoblasts were transfected with a plasmid encoding empty vector, constitutively active p38␣, or p38 as indicated. A plasmid encoding C/EBP mutant LAP-T188A tagged with the FLAG was cotransfected as indicated. After differentiation for 96 h, expression of the plasmids and UBR2 was evaluated by Western blotting. Data were analyzed with ANOVA. *P ⬍ 0.05 vs. vector-only control; † P ⬍ 0.05 vs. single transfection of active p38. B) C/EBP phosphorylation at Thr-188 is critical to UBR2 up-regulation by tumor cell-conditioned medium. C2C12 myoblasts were transfected with empty vector or a plasmid encoding either wild-type LAP or LAP-T188A. After differentiation, the cells were treated with LCM or control medium for 8 h. Expression of the plasmid and UBR2 was evaluated by Western blotting. Data were analyzed with ANOVA. * P ⬍ 0.05 vs. vector-only control; †P ⬍ 0.05 vs. LCM-treated control.
ation, heat shock, and high osmotic pressure (34). Elevated muscle p38 MAPK activity has been observed in a number of procatabolic states in humans, including inflammatory myopathy (35), type 2 diabetes (36), and aging (37). Therefore, the p38 MAPK-C/EBP signaling pathway may also be involved in certain types of non-tumor-induced muscle wasting. C/EBP has complex roles in muscle metabolism. On one hand, C/EBP participates in the regulation of muscle anabolism. It promotes endocrine expression of IGF-1 and insulin (38), and, in turn, insulin suppresses transactivation by C/EBP (39). On the other hand, C/EBP appears a negative regulator of striated muscle mass. In cardiac muscle, reduction of C/EBP results in cardiomyocyte hypertrophy and resistance to cardiac failure on pressure overload (40). C/EBP is activated
Figure 7. C/EBP is critical to UBR2 up-regulation in the muscle of LLC tumor-bearing mice. LLC cells or PBS were injected subcutaneously into the right flank of male C/EBP⫺/⫺ mice or wild-type littermates. In 14 d, UBR2 protein levels in the TA of the mice were analyzed by Western blotting. Representative blot with samples from 3 mice/group is shown. Data were analyzed with ANOVA. *P ⬍ 0.05 vs. PBS-injected wild-type mice; †P ⬍ 0.05 vs. LLC tumor-bearing wild-type mice. MECHANISM OF UBR2 UP-REGULATION BY TUMOR
by such inflammatory mediators as LPS, TNF-a, IL-6, and IL-1 (41) that stimulate muscle catabolism (2) and promote cardiac failure (42). We showed recently that C/EBP mediates tumor-induced muscle catabolism via the up-regulation of atrogin1/MAFbx and possibly additional E3 ligases (10). The present study demonstrates that the transactivation of tumor-induced E3 ligase UBR2 is mediated by C/EBP. The etiology of cancer cachexia is highly complex and involves humoral factors released in response to tumor by various tissues including the brain, adipose tissue, and immune cells (2). Notwithstanding, the catabolic effects of tumor cell-conditioned medium on cultured myotubes demonstrated in the present and previous studies (10, 18) clearly indicate the capacity of tumor cells to directly induce muscle catabolism. The fact that patients with lung, pancreatic, colorectal, or gastric cancer are among those most likely to experience significant loss of skeletal muscle mass with or
Figure 8. Working model of the p38 MAPK-C/EBP signaling pathway-mediated muscle mass loss in cancer cachexia. 2899
without anorexia (43, 44) suggests the possibility that certain types of fast-growing tumors sustain their highlevel protein synthesis by releasing substances to extract amino acids from skeletal muscle, the largest storage of body proteins, via stimulating proteolysis. Therefore, identifying tumor cell-released cachexia-inducing factors that activate the p38 MAPK-C/EBP signaling pathway appears to be a key to understanding the etiology of cancer cachexia. This work was supported by an R01 grant from the U.S. National Institute of Arthritis and Musculoskeletal and Skin Diseases to Y.-P.L. (AR052511). The authors thank Peter Johnson (National Cancer Institute, Bethesda, MD, USA) for sharing the C/EBP⫺/⫺ mice, Jiahuai Han (Scripps Research Institute, La Jolla, CA, USA) for sharing the adenovirus encoding MKK6bE, and David Engelberg (Hebrew University, Jerusalem, Israel) for sharing plasmids encoding the constitutively active mutants of p38 MAPK isoforms.
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