The current proposal grew from studies of the molecular mechanisms by which nandrolone protects against muscle atrophy caused by nerve transection and were stimulated by findings that Notch and Wnt play critical roles in tissue repair after muscle injury. Notch stimulates proliferation of satellite cells, a mesenchymal 'stem' cell capable of proliferating to allow self-renewal, and of producing daughter cells able to commit to the skeletal muscle lineage, form myoblasts, and fuse to form new muscle fibers or contribute to existing ones. Wnt inhibits Notch signaling and stimulates myogenic differentiation. Our preliminary data suggest that denervation activates Notch and Wnt signaling in denervated muscle and that nandrolone terminates Notch signaling while further increasing Wnt signaling. Termination of Notch signaling by nandrolone is associated with increased expression of the Notch inhibitor Numb. A preliminary analysis of the sequence of the Numb promotor suggests that it contains binding sites for transcription factors (TCF/LEF) by which Wnts regulate gene expression through activation and nuclear translocation of the transcriptional regulator ss-catenin, as well as possible androgen receptor response elements; the androgen receptor is a ligand-activated transcription factor that binds to such sites to upregulate gene expression. Our hypothesis is that nandrolone induces upregulation of Numb and Wnt signaling in satellite cells, which is critical to protection against atrophy by nandrolone, and dependent upon upregulation of Numb transcription; upregulation is mediated by the androgen receptor and Wnt signaling. The following specific aims are proposed: 1. Determine the cell type(s) in which nandrolone alters Numb, and signaling by Notch and ss- catenin. Our hypothesis is that nandrolone regulates Numb expression and Notch and ss-catenin- dependent signaling in satellite cells and that effects of nandrolone on Numb expression and ss-catenin- dependent signaling occur in the same cells. In a mouse model of denervation atrophy, we will determine the cell type in which nandrolone upregulates Numb and signaling by ss-catenin by immunohistochemistry. 2. Examine the role of Numb in nandrolone-induced inhibition of Notch signaling and protection against denervation atrophy. Our hypothesis is that upregulation of Numb terminates Notch signaling and is necessary for effects of nandrolone to protect against denervation atrophy. We will use a transgenic mouse in which the Numb gene can be inactivated at a specific time. Experiments will test whether inactivation of the Numb gene blocks effects of nandrolone on Notch signaling and protection against muscle atrophy in a mouse model of denervation atrophy. 3. Characterize molecular mechanisms by which nandrolone upregulates Numb. Our hypothesis is that the Numb promotor contains a cluster of androgen response elements, and thus upregulation of Numb by nandrolone requires binding of the androgen receptor to these DNA sequences. In animal models, we will test the role for Wnt signaling in Numb expression in denervated muscle by either knocking out ss-catenin or overexpressing Wnts or their inhibitors. In cell culture, we will determine whether: binding of nandrolone to the androgen receptor (AR) is needed for upregulation of Numb by nandrolone; AREs within the Numb promotor function to upregulate its transcription; and nandrolone alters Numb mRNA stability.
Muscle loss affects veterans with diverse medical conditions that include stroke, spinal cord injury, COPD, asthma and rheumatological disease. It also affects wounded soldiers during recovery from burns and/or immobilization resulting from their injuries. Knowledge that improves treatments to preserve or restore function of these veterans holds the potential to significantly improve the speed of their recovery, and, possibly, their quality of life and community function and integration. Medications that prevent muscle atrophy or promote restoration of muscle mass after illness or injury are not available. Knowledge regarding molecular and cellular processes that might be targets for future medications is thus highly relevant to veterans.
