The AP-1/CJUN signaling cascade is involved in muscle differentiation: Implications in muscle wasting during cancer cachexia

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1 The AP-1/CJUN signaling cascade is involved in muscle differentiation: Implications in muscle wasting during cancer cachexia Rodrigo Moore-Carrasco a,b, Celia García-Martínez a,sílvia Busquets a, *, Elisabet Ametller a, Esther Barreiro c, Francisco J. López-Soriano a, Josep M. Argilés a a Departament de Bioquímica i Biologia Molecular, Cancer Research Group, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, Barcelona, Spain b Departamento de Bioquímica Clínica e Inmunohematología, Universidad de Talca, Talca, Chile c Muscle and Respiratory System Research Unit, IMIM, CEXS, Universitat Pompeu Fabra, Barcelona, Spain Received 4 November 2005; revised 13 December 2005; accepted 30 December 2005 Available online 5 January 2006 Edited by Vladimir Skulachev FEBS Letters 580 (2006) Abstract The aim of the present study was to investigate a possible role of the AP-1 signaling cascade in the process of wasting associated with cancer cachexia at the level of skeletal muscle. The injection of virus containing the TAM67 protein (a blocker of the AP-1 protein) to the gastrocnemius muscle of tumourbearing rats resulted in a significant recovery of the muscle mass (which is dramatically reduced as a result of tumour burden), therefore suggesting that AP-1 is certainly involved in the signaling associated with muscle protein accretion. In conclusion, the gene therapy approach presented here clearly suggests an important role for AP-1 in muscle signaling during catabolic states. Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: AP-1; c-jun; TAM67; Skeletal muscle; Cachexia 1. Introduction One of the most common manifestations of advanced malignant disease is the development of cancer cachexia. Cachexia occurs in the majority of cancer patients before death and it is responsible for the death of 22% of cancer patients [1]. The abnormalities associated with cancer cachexia include anorexia, weight loss, muscle loss and atrophy, anemia and metabolic alterations [2 4]. The degree of cachexia is inversely correlated with the survival time of the patient and it always implies a poor prognosis [5,6]. Perhaps one of the most relevant characteristics of cachexia is that of asthenia (or lack of muscular strength), which reflects the important muscle waste that takes place in the cachectic cancer patient [3]. Asthenia is also characterized by a general weakness as well as physical and mental fatigue [7]. In addition, lean body mass depletion is one of the main trends of cachexia and it involves not only skeletal muscle but also cardiac proteins, resulting in important alterations in heart performance. Several cytokines have been shown to mimic many of the metabolic abnormalities found in the cancer patient during * Corresponding author. Fax: address: silviabusquets@ub.edu (S. Busquets). Abbreviations: NF-jB, nuclear factor kappa B; AP-1, activator protein 1; MHC, sarcomeric myosin heavy chain; TNF-a, tumour necrosis factor-alpha; PIF, proteolysis-inducing factor; COPD, chronic obstructive respiratory syndrome; GFP, green fluorescent protein cachexia [8]. Among these metabolic disturbances and concerning muscle wasting, the role of tumour necrosis factor-alpha (TNF-a) seems very clear. Thus, administration of TNF-a to rats results in an increased skeletal muscle proteolysis associated with an increase in both gene expression and higher levels of free and conjugated ubiquitin [9 12]. In addition, the in vivo action of TNF-a during cancer cachexia does not seem to be mediated by interleukin-1 (IL-1) [13] or glucocorticoids [14]. Concerning a possible direct action of TNF-a on muscle proteolysis, the presence of both p55 and p75 TNF-a receptors has been described [15 17], and we have demonstrated that the action of the cytokine on the induction of ubiquitin-dependent proteolysis can be direct [12]. Other studies have shown that the ubiquitin-dependent proteolytic system is activated in skeletal muscle in both humans [18] and many experimental models [16,19 21]. Other cytokines, such as IL-1 or interferon-c are also able to activate ubiquitin gene expression. Therefore, TNF-a alone or in combination with other cytokines, seems to mediate most of the changes concerning nitrogen metabolism associated with cachectic states. In addition to the massive muscle protein loss, during cancer cachexia muscle DNA is also decreased, similarly to what is observed in skeletal muscle of chronic heart failure patients suffering from cardiac cachexia [22], this leading to apoptosis measured as DNA fragmentation [23]. Interestingly, TNF-a can mimic the apoptotic response in muscle of healthy animals [24]. In order to define successful approximations for the pharmacological treatment of cachexia, a better knowledge of the different intracellular signaling pathways linked to the muscle wasting process is essential. This is especially important since there is a plethora of mediators that have been proposed as being responsible for the muscle protein abnormalities found in cachexia. From this point of view it is interesting to remark that previous studies have shown an involvement of both nuclear factor kappa B (NF-jB) and activator protein 1 (AP-1) signaling cascades during sepsis [25]. Interestingly, in chronic obstructive respiratory syndrome (COPD) patients (with evident muscle wasting) NF-jB has also been proposed as being involved in the loss of skeletal muscle [26]. Studies from our laboratory have shown that NF-jB does not seem to have a key role in experimental cancer cachexia [27], while AP-1 seems to be involved [28]. Bearing all this in mind, the main objective of the present work was to see if the administrations of an adenoviral load genetically-engineered to express the TAM67 protein (a negative dominant of c-jun/ap-1) was able /$32.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi: /j.febslet

2 692 R. Moore-Carrasco et al. / FEBS Letters 580 (2006) to interfere with muscle wasting in cachectic tumour-bearing rats. Additionally, a second objective of the present investigation was to investigate if the AP-1 signaling cascade was also involved in the intracellular actions of TNF-a in the skeletal muscle cells. 2. Materials and methods 2.1. Animals Male Wistar rats (Interfauna, Barcelona, Spain), of three days of age were used. The animals were maintained at 22 ± 2 C with a regular light dark cycle (light on from 08:00 a.m. to 08:00 p.m.) and had free access to food and water. All animal manipulations were made in accordance with the European Community guidelines for the use of laboratory animals Recombinant adenovirus preparation and administration to animals Recombinant adenoviruses containing the cdna encoding TAM67 dominant negative (AdCMV-TAM67) or the green fluorescent protein (AdCMV-GFP) [29] were used. A 1.3 kb-dna fragment containing the TAM67 dominant negative cdna of human c-jun gene was excised with EcoRI from the psg5-tam67 vector (kindly provided by Dr. M.J. Birrer, Department of Cell and Cancer Biology, National Cancer Institute, Rockville, MD [30]) and subcloned into pac- CMVpLpA downstream of CMV promoter. Plasmid padcmv- TAM67 was used to isolate the corresponding AdCMV-TAM67 virus. Viruses were amplified in 293 cells and concentrated to plaque-forming units (pfu)/ml by centrifugation through a CsCl gradient as described previously [29]. Viruses were aliquoted and stored at 80 C in phosphate-buffered saline (PBS). Viruses were titrated by a serial dilution plaque-assay in noble agar overlaid 293 cells. Approximately pfu of AdCMV-TAM67 or AdCMV-GFP were delivered to newborn (3 4 days) Wistar rats by intramuscular injection in the right or left hind leg gastrocnemius muscle, and the right hind leg was injected the same volume of PBS. Animals treated with AdCMV- TAM67 or AdCMV-GFP were analyzed for transgene expression in muscle. Only animals with detectable transgene expression were used for further analysis Tumour inoculation Rats were divided into two groups, namely controls and tumour hosts. The latter received an intraperitoneal inoculum of AH- 130 Yoshida ascites hepatoma cells obtained from exponential tumours [31]. On day 4 after tumour transplantation, the animals were weighed and anaesthetized with an i.p. injection of ketamine/xylazine mixture (3:1) (Imalgene Ò and Rompun Ò, respectively). Tissues were rapidly excised, weighed, and frozen in liquid nitrogen Cell culture and transduction with adenovirus The murine myoblasts C2C12 [32] were cultured in Dulbecco s modified Eagle s medium (DMEM) supplemented with 10% fetal bovine serum, 20 mm glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (growth medium, GM). To induce differentiation cells at 80 90% confluence were shifted to DMEM supplemented with 2% fetal bovine serum (differentiation medium, DM). For transduction experiments, C2C12 cells were plated in GM for 24 h at a density of cells/35-mm plate. Cells were then infected with the adenoviruses [29]. After transduction, cells were transferred in DM and exposed to TNF-a. Construction and use of recombinant adenoviruses containing the TAM67 dominant negative (AdCMV-TAM67) or the cdna encoding a GFP (AdCMV-GFP). Gene delivery to muscle cultures was achieved by exposing myoblasts, to the virus for 2 h at a multiplicity of infection of 100 [33] Western blotting For Western blots, protein extracts from tissues were prepared in standard lysis buffer (50 mm Tris, ph 7.4, 0.25 mm sucrose and 5 mm EDTA, ph 8.0, and a cocktail of protease inhibitors) and analyzed using anti-myod (Santa Cruz Sc-760), anti-mhc (Hybridoma Bank MF20), anti-ciclin D1 (Santa Cruz Sc-8396), anti-c-jun (Oncogene PC06), anti-a-tubulin (Sigma T5168), or anti-gfp (Clonthech CT6833) antibodies. 10 lg of total protein extract were loaded for each sample. Specific proteins were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and a chemiluminescence kit. The sizes of proteins were estimated by using protein molecular-mass standards. Protein bands were quantified by scanning densitometry (Phoretix 1D International Ltd., England) In vivo detection of GFP expression A Leica fluorescence stereomicroscope (model LZ12) equipped with a 50-W mercury lamp was used for high-magnification imaging. Selective excitation of GFP was produced through a D425/60 band-pass filter and 470 DCXR dichroic mirror. Emitted fluorescence was collected through a long-pass filter GG475 (Chroma Technology, Brattleboro, VT). At different times after gene transfer, animals were anaesthetized and photographed of region of interest were taken either directly through the skin or after skin removal [34,35] Biochemicals These were all reagent grade and obtained either from Roche S.A. (Barcelona, Spain) or from Sigma Chemical Co. (St. Louis, MO, USA) Statistical analysis Statistical analysis of the data was performed by means of the Student s t test. 3. Results and discussion Cancer cachexia is a complex syndrome characterized by weight loss, anorexia and profound disturbances in metabolism which inflict serious muscle wasting. Several experimental models have been used to study muscle wasting during cancer. The rat ascites hepatoma Yoshida AH-130 is a suitable model system to study the mechanisms involved in the establishment of cachexia, because its growth causes rapid and progressive loss of body weight and tissue waste in the host, particularly in skeletal muscle [31]. Unfortunately, there seems to be a plethora of mediators involved in the activation of muscle protein degradation during cancer cachexia. Thus, several tumour compounds have been proposed as activators of protein breakdown. Proteolysisinducing factor (PIF) has been involved in the activation of muscle wasting in an experimental mouse model of cancer cachexia [36]. Additionally, cytokines, TNF-a and IL-6 in particular, have also been reported as having a role in wasting in skeletal muscle during cancer [37 40]. Changes in hormone concentrations and/or sensitivity (insulin, glucocorticoids) may also be involved in wasting [41 43]. Taking all of this into consideration, the design of an effective therapeutic strategy is rather complicated due to the number of different mediators. Bearing this in mind, it may be more effective to see if the therapeutic strategy may be directed to an intracellular common target, since some of the compounds mentioned seem to share similar intracellular signaling pathways. However, few studies have considered the intracellular signaling pathways associated with muscle wasting. Penner et al. [25] reported that during experimentally-induced sepsis there was an activation of both NF-jB and AP-1 signaling pathways. In humans the muscle wasting associated with the COPD seems to be also related with activation of NF-jB associated with degradation of IjB alpha [26]. Although some studies seem to indicate a role for NF-jB in muscle wasting [44], previous results from our laboratory

3 R. Moore-Carrasco et al. / FEBS Letters 580 (2006) indicate that in experimental cancer cachexia there is an activation of AP-1 while NF-jB does not seem to be involved [27,28]. We therefore decided to investigate if blockage of the AP-1 signaling cascade could be an effective therapeutic approach for the treatment of cancer cachexia. Fig. 1A shows that the infection with the adenovirus was highly effective. Indeed, the photographs show that both in gastrocnemius and soleus, the presence of the GFP was clearly manifested 30 days following injection of the virus. It has to be pointed out that, although the viral injection was performed at the gastrocnemius muscle, there seems to be some spreading of the viral load to adjacent muscles, such as soleus. Fig. 1B shows a Western blot indicating that the proteins (TAM67 and GFP) were expressed only in the gastrocnemius muscles where they were injected, right and left for AdCMV-TAM67 and AdCMV-GFP, respectively. As shown in Fig. 2A, the implantation of the tumour resulted in a significant decrease in gastrocnemius mass (12%). This result is in agreement with previous results from our research group using the same experimental model [13,14,45]. Interestingly, the administration of the TAM67-containing adenovirus resulted only in a 5% decrease in gastrocnemius mass, this being statistically different from the decrease observed in the control animals injected with AdCMV-GFP, where the decrease in muscle mass was around 10%. It can therefore be speculated that the in vivo blockage of the AP- 1/c-jun signaling cascade is able to partially block muscle mass loss. It has to be pointed out that, since the blockage of the AP-1 transcription factor results only in a partial reversal of the muscle mass, other transcription factors such as NF-jB are likely to also be involved [44,46,47]. We then decide to investigate if the effects of AdCMV-TAM67 on muscle mass were correlated with changes in the transcription factor MyoD. As can be seen in Fig. 2B, the implantation of the tumour resulted in a significant decrease of MyoD content (22%). Very interestingly, the injection of the AdCMV- TAM67 carrying virus neutralized the decrease in MyoD content and the presence of the tumour did not seem to significantly affect the content of the transcription factor in the tumour-bearing animals. In previous studies involving experimental models of cancer cachexia, we have described that cytokines, TNF-a in particular, may be the mediator(s) triggering muscle protein breakdown during catabolic states. In fact, both in vivo [9] and in vitro [48], it has been described that this cytokine is responsible for activating myofibrillar protein degradation [49,50]. Bearing all this in mind, we decided to investigate whether AP-1 was involved in the TNF-a signaling cascade in skeletal muscle cell cultures. We therefore exposed C2C12 cells both control and adenovirus-infected in culture to the action of the cytokine. As can be seen in Fig. 3, the viral infection of the muscle cell cultures was successful, the photograph showing the GFP protein fluorescence (Fig. 3A). Fig. 3B depicts a Western blot of the TAM67, thus confirming that the protein was present in the transfected cells. The data presented in Fig. 4 clearly show that the addition of the TNF-a to the cell cultures resulted in a significant decrease Fig. 1. Analysis of efficiency of the in vivo adenoviral transduction using fluorescence microscopy and Western blotting. (A) Fluorescence microscopy images of skeletal muscle from rats 30 days after injection of the virus. (B) Western blot of gastrocnemius muscles injected with AdCMV-TAM67 (TAM67) (injected in right thigh muscle) or AdCMV-GFP (GFP) (injected in left thigh muscle). L: left muscle, R: right muscle. Fig. 2. Gastrocnemius muscle weight (A) and MyoD content (B) in control and tumour-bearing rats treated with AdCMV-TAM67. For full details, see Section 2. The results are means ± S.E.M. of a minimum of 4 animals for group. (1) Non-infected (N.I.). (2) AdCMV- TAM67 (TAM67). (3) AdCMV-GFP (GFP). Statistical analysis of the results (Student s t test): control vs. tumour **,P ; AdCMV- TAM67 (TAM67) vs. AdCMV-GFP (GFP), P both in the amount of MyoD (50%) (Fig. 4A) and sarcomeric myosin heavy chain (MHC) (80%) (Fig. 4B). These results agree with previous work from our laboratory using the same cell culture system [48]. Very interestingly, the TAM67 expressing cells showed a resistance to the cytokine effects on both MyoD and MHC content. Several conclusions can be drawn

4 694 R. Moore-Carrasco et al. / FEBS Letters 580 (2006) Fig. 3. Analysis of the in vitro adenoviral transduction using fluorescence microscopy and Western blotting. (A) Fluorescence microscopy image of C2C12 differentiating myoblasts 72 h after infection with AdCMV-GFP. (B) Western blot of C2C12 differentiating myoblasts 72 h after infection with AdCMV-TAM67. from these results. (1) TNF does indeed act catabolically in the cell culture system, clearly decreasing the myofibrillar protein content; (2) a good correlation of changes exist between the transcription factor (MyoD) and the myofibrillar protein content (MHC), since very similar changes affect both of the proteins; (3) the effects of the cytokine on the protein content involve the AP-1 signaling cascade; (4) the AP-1 transcription may be involved in muscle cell differentiation since the simple expression of the TAM67 protein (AP-1 blockade) increases the amount of MHC (a protein clearly associated with skeletal muscle differentiation) by 300% (Fig. 4B). In fact, in order to confirm this point from a different angle, we decided to measure the content of cyclin D1 (a protein clearly associated with proliferation). As can be seen in Fig. 5, the addition of TNF-a to the cell cultures resulted in a significant increase both in control (non-transduced) and control (AdCMV-GFP-transduced) (30% and 70%, respectively) in cyclin D1 content (in control both infected and non-infected), suggesting that the action of the cytokine promotes proliferation as opposed to differentiation. Very interestingly, the presence of the TAM67 protein completely abolished the effect of TNF-a on cyclin D1 content. In fact, the observed effect of the cytokine on the TAM67-expressing cells was completely opposite: TNF-a treatment actually resulted in a decreased cyclin D1 content when the AP-1 signaling cascade was blocked (Fig. 5). In summary, the data presented here clearly support a role for the AP-1 signaling cascade in muscle wasting associated with cancer. In addition, it may be suggested that, at least for TNF-a, AP-1 also seems to be involved in the course of events. However, more studies are necessary to find out if AP-1 is also involved in the intracellular signaling of other proposed muscle wasting mediators such as IL-6 or tumour factors, such as PIF. These studies are particularly important since they may lead to the possibility of introducing drugs able to block the referred signaling cascade which may, in future, constitute a promising therapeutic strategy for the successful treatment of cancer cachexia and muscle wasting associated with other catabolic states. Fig. 4. MyoD and MHC content in control and tumour-bearing rats treated with AdCMV-TAM67. (A) MyoD content (B) MHC content. For full details, see Section 2. The results are means ± S.E.M. of a minimum of 4 animals for group. Non-infected (N.I.), AdCMV- TAM67 (TAM67), AdCMV-GFP (GFP). Statistical analysis of the results (Student s t test): control vs. TNF-a \, P ; \\, P ; AdCMV-GFP vs. AdCMV-TAM67, P Fig. 5. Cyclin D1 content in control and tumour-bearing rats treated with AdCMV-TAM67. For full details, see Section 2. The results are means ± S.E.M. of a minimum of 4 animals for group. Non-infected (N.I.), AdCMV-TAM67 (TAM67), AdCMV-GFP (GFP). Statistical analysis of the results (Student s t test): control vs. TNF-a \, P ; AdCMV-GFP vs. AdCMV-TAM67, P

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