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P&F Grant Awards

Year 3


Grant # 5

The Roles of CD47 in muscle homeostasis and mitochondrial regulation and in skeletal homeostasis.

PI: William Frazier, Ph.D.,

 

Specific Aims

CD47 is a widely expressed receptor for the matricellular protein thrombospondin-1 (TSP1) and for the immunoregulatory counter-receptor SIRPα (signal inhibitory regulatory protein-α) (Brown and Frazier, 2001). While mice genetically knocked out for either TSP1 or for CD47 initially displayed no remarkable phenotypes, my group along with several collaborators, have discovered a number of interesting musculoskeletal phenotypes. With Dr. Katherine Weilbaecher’s group we found that both TSP1 and CD47 play a role in bone homeostasis (Uluckan et al., 2009). In knockouts of either TSP1 or CD47, bones have increased trabecular volume and bone mineral density. CD47 null bone marrow macrophages are severely impaired in their ability to differentiate into osteoclasts (OCs) in vitro, but increasing RANKL levels or reducing NO levels with L-NAME treatment permits their differentiation into OCs. Furthermore, CD47 null mice are protected from tumor metastasis to bone in a model of left ventricle administration of melanoma cells. This data supports the “vicious cycle” model of bone destruction by tumors, however all cells in the host animal are lacking CD47 and therefore a specific role for CD47 on OCs cannot be assumed; recent data suggests that CD47 influences lineage decisions of mesenchymal stem cells as well (Uluckan, Weilbaecher and Frazier, in preparation).

In addition to its role(s) in bone, CD47 affects mitochondrial biogenesis/turnover in both skeletal and cardiac muscle. CD47 limits both the cGMP and cAMP signaling pathways, both of which can regulate mitochondrial biogenesis. Therefore we screened expression of mitochondria-related genes in several tissues of young CD47-null and WT mice. Only skeletal muscle had markedly elevated levels of such genes. Skeletal muscle from CD47 knockouts displayed a markedly increased volume of mitochondria compared to WTs and fast twitch muscle had undergone fiber type switching to a slow twitch phenotype as occurs with exercise training and calorie restriction. Consequently, young CD47-null mice have twice the exercise endurance of wild type mice. The CD47 nulls utilize less oxygen and produce dramatically less ROS than WTs. When we profiled gene expression in tissues as a function of age, we found that, by 1 year of age, CD47 null skeletal muscle mitochondrial density had declined to WT levels. To our surprise, CD47 null hearts of aging mice develop dramatically increased mitochondrial density that correlates with improved heart function. Thus CD47 plays a role in both skeletal and cardiac muscle function as well as in bone physiology.

In order to sort out the complex phenotypes of CD47-nulls in muscle and bone, each of which contain multiple cell types derived from diverse precursor and stem cell lineages, we propose to generate mice harboring a floxed CD47 gene that, when crossed with mice harboring Cre recombinase under the control of tissue-specific promoters, will allow us to delete CD47 expression in a cell- or tissue-specific fashion. The knockout mouse project (KOMP) has successfully produced ES cells harboring a floxed CD47 gene. We will use all three components of the musculoskeletal core in the following Specific Aims:

  1. Utilize the mouse genetics component of the musculoskeletal core to generate genetically targeted mice harboring the floxed CD47 gene. Floxed CD47 ES cells have been ordered from KOMP.
  2. To test the functionality of the floxed CD47 gene, floxed CD47 mice will be mated with LysM promoter-Cre mice to disrupt CD47 expression in myeloid cells, including osteoclasts. The LysM-Cre mice are available from the Weilbaecher lab (Morgan et al., 2009). We will access the in situ analysis core to help characterize the tissue-specific CD47 knockouts for CD47 expression, histology and histomorphometry of bone.
  3. Bone structure and density (DEXA, microCT) as well as strength testing of the conditional knockouts will be performed with the structure and strength core.
Once the floxed CD47 line is established and verified, we will mate the mice with other promoter-Cre lines to generate tissue-specific CD47 nulls in mature osteoclasts, osteoblasts, platelets, skeletal, smooth and cardiac muscle, and reticulocyte/erythrocytes. Using the CD47 global knockouts, we and others have obtained data implicating CD47 in functions of all of the above cell and tissue types. Only targeted knockouts can test the cell or tissue autonomous function of CD47. Therefore, this pilot project will lay the essential groundwork for many additional NIH-funded projects to come, thereby fulfilling the purpose of this pilot program.