Mechanisms and Impact of Osteoblast "Citration" on Skeletal Mineralization and Global Citrate Homeostasis - ABSTRACT The body maintains an adequate balance between citrate availability and elimination, depending on physiological needs and determined by diet, renal clearance, cell metabolism and bone remodeling. Citrate is used by all aerobic organisms to produce usable chemical energy and is present in bone at strikingly high concentrations (1-5 wt%). In fact, two independent studies using high resolution NMR to model the citrate molecule within the apatite crystal suggest that the degree of citrate incorporation, as well as its spatial orientation within the mineral structure, is critical for maintaining favorable biomechanical properties. These observations prompt several fundamental questions that form the basis for this proposal: 1) What is the mechanism for citrate delivery to bone?; 2) How does the partitioning of citrate in bone influence global citrate handling?; and 3) how is this partitioning regulated? In preliminary studies, we demonstrate functional expression of a membranous extracellular Na+/citrate cotransporter, Solute Carrier Family 13 Member 5 (Slc13a5), in mineralizing osteoblasts. Interference of SLC13A5-mediated citrate transport, either genetically or pharmacologically, disrupts osteoblast mediated mineral nucleation. Mice lacking Slc13a5 show increased serum and urinary citrate levels, reduced bone volume and quality, and defects in tooth enamel, pathological features similar to those seen in humans with mutations in SLC13A5. Intriguingly, metabolic flux analysis revealed striking elevations in 13C-Glucose-derived 13C-Citrate (m+2) in apatite deposited by Slc13a5 null osteoblasts which was allocated to increased mitochondrial citrate production and export. Moreover, we found that Slc13a5 expression was strongly regulated by the calciotropic parathyroid hormone (PTH). These findings suggest the existence of an osteoblast specific mechanism that controls both the production and delivery of citrate to bone as well as systemic citrate availability. Specifically, we postulate that the membrane citrate transporter SLC13A5 senses extracellular citrate concentrations and enables the osteoblast to adjust its endogenous citrate production when extracellular citrate levels drop or in response to calcium regulating hormones such as PTH. Three aims were developed to assess our new metabolic pathway downstream of SLC13A5 in a human disease model using hiPSCs and primary teeth derived from patients with SLC13A5 disease and to define the role of SLC13A5 in citrate partitioning between blood and bone in physiological conditions or in response to PTH. As a young scientist, my ultimate goal is to conduct productive research that provides scientific insights into skeletal mineralization and the integration of these mechanisms in general physiology. My career development plan has been tailored toward this goal with solid mentorship, collaborations, and training opportunities. In conjunction with institutional support, I am confident that the studies/activities outlined in my application will help build upon my existing skillset and facilitate my transition into an independent investigator.
