Browsing by Subject "Myogenesis"
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Item AP-1 signaling in skeletal muscle differentiation(Texas Tech University, 2001-05) Icli, BasakHeart 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.Item Mechanistic Analysis of SRF and the Myocardin Family of Coactivators During Muscle Development(2005-12-20) Li, Shijie; Olson, Eric N.The precise mechanism of how specification and differentiation of different muscle types are controlled by a large number of transcription factors has been a long-standing question in developmental biology. Using animal models with tissue-specific deletions of various transcription factors, coupled with biochemical studies, the molecular mechanisms regulating muscle development and growth are being elucidated. Serum response factor (SRF), a muscle-enriched transcription factor, activates the expression of numerous muscle genes by recruiting a variety of partner proteins. The function of SRF in each muscle type in vivo is clouded by the fact that SRF mutant mice die before gastrulation without the formation of mesoderm. Generating a tissue-specific deletion of the SRF gene, I found that SRF is required for skeletal muscle growth and maturation. Myocardin was identified as a cardiac and smooth muscle-specific transcriptional coactivator of SRF. Mice lacking myocardin die during early embryogenesis due to cardiovascular defects, which are caused by the failure of vascular smooth muscle to differentiate. Together with the data that overexpression of myocardin in non-muscle cells can activate the smooth muscle gene program, we demonstrate that myocardin is both required and sufficient for smooth muscle differentiation. Two Myocardin Related Transcription Factors, referred to as MRTF-A and B, which also interact with SRF and stimulate its transcriptional activity, are expressed in numerous embryonic and adult tissues, implying their potential to modulate SRF target genes in a wide range of tissues. Consistent with the role of SRF during skeletal muscle development, a dominant-negative form of MRTF-A interferes with skeletal muscle development in transgenic mice. To further elucidate MRTF-A's function, I generated MTTF-A mutant mice by gene homologous recombination. Female MRTF-A mutant mice fail to nurture their offspring due to mammary defects. While milk is produced at a normal level, mammary myoepithelial cells, which are similar to smooth muscle cells and required for milk ejection, fail to differentiate and undergo programmed cell death during lactation. Taken together, these data indicated that SRF regulates specification or maturation of different muscle types by interacting with various members of the myocardin family of coactivators.Item Transcription factor involvement in the rat PNS-derived stem cell line family, RT4(Texas Tech University, 1998-05) Reinhart, Adam JThe long-term objectives of this project were twofold: (1) to identify transcription factors (both novel and previously identified) which may influence cell fate and/or maturation in the nervous system, and (2) to investigate the role(s) that these transcription factors may play in either the cell-fate decisions and/or maturation of an in vitro model of peripheral neurogenesis called RT4. In the first phase of this project, five POU-domain genes (Oct-1, Oct-2, Bm-2, Bm- 5 and Tst-1/SCIP), as well as REST/NRSF (a repressor of neuronal-specific gene expression) were shown to be expressed, and their expression levels characterized in the RT4 cell line family. We also reported that 4 candidate transcription factors (Bm-1, Bm-3, Bm-4 and MASH-1) were not expressed in the RT4 cell line family. The second phase of this project involved the elucidation of the possible role(s) of Tst-1/SCIP and REST/NRSF in the RT4 cell line family. We examined whether Tst-1/SCIP influenced the conversion of RT4 stem cells to derivative cell types. The conversion frequency was essentially unchanged when transfected with wild-type Tst-1/SCIP, or a dominant-negative version of Tst-1/SCIP, or pcDNA3 vector alone. Although the conversion frequency among transfection groups (i.e., Tst-1/SCIP; Tst-1/SCIP AN; pcDNA3 vector alone) groups was approximately the same to all of the derivative cell types, the total number of stably transfected colonies within the Tst-1/SCIP transfection group was considerably less than the other transfection groups. The transcription factor REST/NRSF was examined in terms of its possible role(s) in regulating maturation-specific gene expression. Although we were able to demonstrate that the REST/NRSF-mediated repression of an RE 1-CAT reporter was at least partially relieved, we were unable to detect the de novo expression of a set of maturation-specific genes in RT4-B8, which would expect to be expressed if the cell line had been stimulated to mature.