Kim C. Mansky - US grants

DESCRIPTION The severe bone phenotype in microphtalmia (MITF) mutant mice indicates that the helix-loop-helix (HLH) zipper transcription factor encoded by the microphthalmia (MITF) gene has significant role in terminal differentiation of multi-nucleated osteoclasts. In addition to providing a very interesting system to study developmentally regulated gene expression in a mammalian system, studying the MITF gene may have direct impact on human disease. In particular, osteoporosis in post- menopausal women and the osteolytic bone destruction and hypercalcemia that occurs in patients with multiple myeloma are examples of clinical conditions where this research may have potential impact. The long term goal of this grant are to determine, at the molecular level, the function of the MITF protein in normal osteoclast biology. The specific aims of the proposal are: 1. To determine if MITF is a target of Erk-2 in osteoclasts 2. To determine if MITF is a target of ERK-2 in osteoclasts. 2. To determine if MITF is a target of Jun kinase (JNK) in osteoclasts. These studies will be performed in three experimental systems: heterologous cell lines, primary osteoclast-like cells in vitro and in vivo in mouse transgenic models.

[unreadable] DESCRIPTION (provided by applicant): Microphthalmia-associated transcription factor (Mitf) and Tfe3 are basic helix-loop-helix leucine zipper transcription factors that are expressed in osteoclasts. We have demonstrated that Mitf and Tfe3 colloborate to activate genes important for osteoclast differentiation. Evidence that Mitf and Tfe3 are necessary for osteoclast differentiation is demonstrated by the fact that mice null for expression of Mitf and Tfe3 are osteopetrotic. Using a yeast two hybrid screen, we identified Cdc25C-associated kinase 1 (C-TAK1) and POH1, a component of the proteasome lid, as interacting with the amino terminus of Mitf. This proposal attempts to 1) determine how Mitf is translocated to the nucleus in osteoclasts and 2) to identify proteins that affect Mitfs stability in osteoclasts. To define the interaction between C-TAK1 and Mitf (aim 1), we will determine which amino acid residue of Mitf is phosphorylated by C-TAK1, determine which Mitf amino acid residues are critical for binding to 14-3-3 proteins and determine what effect C-TAK1 and 14-3-3 interaction has on Mitfs cellular location. 14-3-3 are a family of proteins that recognize specific phosphorylated serines and bind to and sequester a protein in the cytoplasm. To determine the effect of Mitf and POH1's interaction (aim 2), we will determine if Mitf's stability is affected by CSF-1 and RANKL signaling, map the region of Mitf that interacts with POH1 and determine the effect of POH1's interaction with Mitf on Mitfs activation of osteoclast target genes. By understanding how Mitf activates ostoeclast specific genes, drug targets may be suggested that will lead to therapies to control the differentiation and activation of osteoclasts. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Novel approaches to reduce osteoclastic bone loss are needed for a number of exceedingly common clinical conditions including osteoporosis, metastatic bone disease, periodontitis, and arthritis; conversely, strategies to increase osteoclast function are necessary to correct the lack of bone resorption in conditions including osteopetrosis. To improve our ability to clinically manipulate osteoclast activity will require a deeper understanding of the molecular mechanisms that govern their physiology. We have identified histone deacetylase 7 (HDAC7) as a negative regulator of osteoclast differentiation that has potential implications for the development of new therapies. While inhibition of other HDACs impairs osteoclastogenesis, preliminary studies reveal a unique function for HDAC7 in osteoclasts. Suppression of HDAC7 enhances their formation, while their formation is impaired by overexpression of HDAC7. Using the LysM-Cre mouse, which targets osteoclasts, we have preliminary data demonstrating that at 3 months of age HDAC7-null mice are osteopenic due to enhanced osteoclastogenesis. Further data indicate that these effects are mediated through RANKL-regulated interactions between HDAC7 and the MITF transcription factor. These results suggest that reduced HDAC7 activity in osteoclastic cells may contribute to pathological bone loss, whereas stimulation of HDAC7 might represent a novel strategy to clinically reduce bone loss. However, the current incomplete understanding of HDAC7's function in osteoclasts limits the rational development of such diagnostic or therapeutic approaches. Our central hypothesis is that HDAC7 is a negative regulator of osteoclast differentiation and functions by repressing the activation of MITF and PU.1 (and potentially other transcription factors). RANKL signaling through the p38 MAP kinase pathway disrupts these repressive interactions, enabling efficient osteoclast gene expression and subsequent differentiation. We will test this hypothesis with three specific aims: 1) Characterize the in vivo phenotype and cellular effects of conditional knockout of HDAC7 in osteoclast progenitors; 2) Characterize the molecular mechanisms by which HDAC7 regulates osteoclast differentiation; and 3) Determine the mechanism and biological significance of RANKL regulation of MITF/PU.1-HDAC7 interaction. Completion of these aims will significantly increase our knowledge concerning a unique regulatory pathway in osteoclasts, advance the search for improved therapeutic strategies for aberrant bone loss and ultimately lead to be better clinical outcomes.

The Minnesota Craniofacial Research Training Program is a direct response to PAR-15- 101, Institutional Training for a Dental and Craniofacial Research Workforce (T90/R90), and the national need to train the next generation of biomedical scientists to work on significant problems in craniofacial, dental, and oral health research. The mission of the program is to engage trainees in novel, mentored research that is fundamental to biology and human health, and translational research that expands the frontiers and scope of craniofacial, dental, and oral health. To foster our mission and ensure strong mentorship and inter-disciplinary research training, a non-hierarchical and highly consultative administrative structure is in place. Trainees engage in research training opportunities with groups of experienced, dedicated and well-supported mentors in Neuroscience, Microbiology and Immunology; Cancer Biology; Developmental Biology, Molecular Genetics, and Stem Cells; Biophysical Sciences; Nanotechnology, Materials Sciences, and Tissue Engineering; and Genomics, Proteomics, Structural Biology, and Computational Biology. Trainee T90 programmatic options include DDS/PhD (DSTP), predoctoral PhD, postdoctoral fellows, and R90 postdoctoral PhD for international dental clinicians. The Minnesota Craniofacial Research Training program builds upon our considerable training experience, outstanding applicant pools and partnerships, new initiatives to recruit and retain DSTP fellows, and appreciation for creative mentored research training. Our alumni work at the state-of-the-art to expand the frontiers of biology and craniofacial, dental, and oral health research.

This project will study bone loss in in microgravity. Bone loss occurs during spaceflight, long-term bedrest and osteoporosis. Utilizing an environment that amplifies the rate of bone loss, such as low earth orbit and ground-based microgravity simulation, will accelerate our understanding of the metabolic mechanisms involved in bone maintenance. Increased understanding of the mechanics of bone loss will help identify affected mechanism(s) and pathways that can be addressed to mitigate bone loss in reduced gravity environments as well as on earth. Educational benefits from this investigation include incorporating the results into the training of undergraduate and graduate students for research careers as basic scientists or clinical investigators. The research will also be a component of the Multicultural Summer Research Opportunities Program.

A recent retrospective multi-omics analysis of hundreds of biological samples flown in space identified mitochondrial dysfunction as the probable root cause for most of the observed physiological changes in a microgravity environment which includes the loss of bone and muscle mass. Monocultures of anabolic cells, i.e., osteoblasts (OB’s), and catabolic cells, i.e, osteoclasts (OC’s), and an OB/OC co-culture will be grown in a collagenous carrier combined with hydroxyapatite as a slurry. These cultures will be maintained by slow perfusion of media flowing through microphysiological chips. Once initiated, the experimental apparatus is autonomous and will be maintained at 37 degrees Celsius/5% CO2 for up to 21 days. The proposed osteogenic microphysiological system, will allow real-time non-contact monitoring of oxygen and pH within the chip to yield OCR (Oxygen Consumption Rate) and ECAR (Extracellular Acidification Rate) metabolic data. These measurements will permit the evaluation of cellular response, i.e., mitochondrial function, to environment or interventions much sooner. At the conclusion of the experiments cell cultures will receive RNAprotect for later transcriptomic analyses. Spent cell media will be analyzed by spectroscopy to determine if glucose metabolic pathways change in microgravity. Utilizing an environment that amplifies the rate of bone loss, such as low earth orbit, or microgravity simulation utilizing a unique magnetic levitation apparatus, provides a setting to accelerate our understanding of underlying genomic and metabolic mechanisms. Validation of a ground-based model of spaceflight may permit a platform to test interventions potentially valuable to the development of therapies beneficial to the treatment of human disease resulting from skeletal unloading, such as paraplegia, bedrest, and metabolic osteopenia/osteoporosis. This work is jointly funded by the Biomechanics and Mechanobiology program and the Engineering of Biomedical Systems program.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.