Graphical AbstractHighlights d Skeletal stem cells increase glutamine metabolism during osteoblast differentiation d Glutamine metabolism regulates osteoblast and adipocyte specification d Mice unable to metabolize glutamine have less bone and increased marrow fat d Amino acid transaminase-derived a-ketoglutarate is critical for SSC proliferation SUMMARY Skeletal stem cells (SSCs) are postulated to provide a continuous supply of osteoblasts throughout life. However, under certain conditions, the SSC population can become incorrectly specified or is not maintained, resulting in reduced osteoblast formation, decreased bone mass, and in severe cases, osteoporosis. Glutamine metabolism has emerged as a critical regulator of many cellular processes in diverse pathologies. The enzyme glutaminase (GLS) deaminates glutamine to form glutamate-the ratelimiting first step in glutamine metabolism. Using genetic and metabolic approaches, we demonstrate GLS and glutamine metabolism are required in SSCs to regulate osteoblast and adipocyte specification and bone formation. Mechanistically, transaminasedependent a-ketoglutarate production is critical for the proliferation, specification, and differentiation of SSCs. Collectively, these data suggest stimulating GLS activity may provide a therapeutic approach to expand SSCs in aged individuals and enhance osteoblast differentiation and activity to increase bone mass.Conceptualization, C.M.K.; Investigation, Y.Y., H.N., L.
Cellular differentiation is associated with the acquisition of a unique protein signature which is essential to attain the ultimate cellular function and activity of the differentiated cell. This is predicted to result in unique biosynthetic demands that arise during differentiation. Using a bioinformatic approach, we discovered osteoblast differentiation is associated with increased demand for the amino acid proline. When compared to other differentiated cells, osteoblast-associated proteins including RUNX2, OSX, OCN and COL1A1 are significantly enriched in proline. Using a genetic and metabolomic approach, we demonstrate that the neutral amino acid transporter SLC38A2 acts cell autonomously to provide proline to facilitate the efficient synthesis of proline-rich osteoblast proteins. Genetic ablation of SLC38A2 in osteoblasts limits both osteoblast differentiation and bone formation in mice. Mechanistically, proline is primarily incorporated into nascent protein with little metabolism observed. Collectively, these data highlight a requirement for proline in fulfilling the unique biosynthetic requirements that arise during osteoblast differentiation and bone formation.
Osteoblasts are the principal bone forming cells. As such, osteoblasts have enhanced demand for amino acids to sustain high rates of matrix synthesis associated with bone formation. The precise systems utilized by osteoblasts to meet these synthetic demands are not well understood. WNT signaling is known to rapidly stimulate glutamine uptake during osteoblast differentiation. Using a cell biology approach, we identified two amino acid transporters, Slc7a7 and Slc1a5, as the primary transporters of glutamine in response to WNT. Slc1a5 mediates the majority of glutamine uptake, whereas Slc7a7 mediates the rapid increase in glutamine uptake in response to WNT. Mechanistically, WNT signals through the canonical/β-catenin dependent pathway to rapidly induce Slc7a7 expression. Conversely, Slc1a5 expression is regulated by the transcription factor ATF4 downstream of the mTORC1 pathway. Targeting either Slc1a5 or Slc7a7 using shRNA reduced WNT induced glutamine uptake and prevented osteoblast differentiation. Collectively these data highlight the critical nature of glutamine transport for WNT induced osteoblast differentiation.
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