AP-1 signaling in skeletal muscle differentiation
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Heart failure is the final common pathway of most of the primary cardiovascular diseases, including hypertension, diabetes, cardiomyopathy, and valvular and congenital malfunctions. Most treatments slow the course of these primary diseases, but do not abolish them. Despite the significant progress in prevention and therapy of heart disease, treating patients with heart failure after myocardial infraction remains a major therapeutic challenge. Adult cardiomyocytes cannot regenerate after injury. One of the most fascinating and potentially beneficial goals in the field of gene therapy for cardiovascular disease would be genetic manipulation leading to regeneration of myocytes after myocardial infraction. A potential strategy is to repopulate infracted areas of the myocardium with skeletal myocytes, which have been genetically engineered to reproliferate. In muscle cells, as in other cell types, the decision to divide or differentiate is determined by a balance of opposing cellular signals. During skeletal muscle differentiation (myogenesis), mononucleated proliferating myoblasts stop dividing, coordinately activate muscle specific gene expression and fuse into multinucleated myotubes. It has been shown that during hypertrophy the activation of a fetal gene called skeletal alpha- actin (SkA) is associated with the AP-1 induction (Paradis et al., 1996). The AP-1 transcriptional factor is composed of dimers between Jun and Fos proteins. Thus characterization of the function of AP-1 in muscle differentiation may help to develop a new gene therapy technique for cardiovascular diseases. Our hypothesis is that the AP-1 activity plays an important role in directing skeletal muscle cells to remain in a proliferative state. As a first step towards this goal, we engineered two different cell lines (C2C12 and SOLS) to generate the AP-1 reporter cell lines. The engineered cell lines are called C2/3XTRE and SOL8/3XTRE. The AP-1 activity during both proliferation and differentiation was measured in these engineered cell lines. We then used a dominant negative c-Jun molecule to down-regulate the AP-1 activity in the engineered cell lines and in HeLa cells (as a control of the results obtained by using the SOL8/3XTRE and C2/3XTRE). The results of this study can be summarized as follows: 1. AP-1 activity decreases as the growth factors in the growth medium decreases. 2. Expression of a dominant negative c-Jun (A169-cjun) induces myoblast differentiation in high serum. 3. AP-1 activity is a necessary signaling component directing myoblasts to remain in a proliferative state. 4. The AP-1 transcription factor is a promising target for gene therapy in muscle cells. Suggested future work is the generation of a stable AP-1 reporter cell line, which should help to analyze both the AP-1 function and its components in a detailed manner. This will allow controlled expression of the gene of interest.