Glutaminase acts in osteoblasts to regulate bone formation
Authors: Karner Courtney Michael , Yu Yilin , Tang Jackie 
Published: November 2023

Osteoblasts are secretory cells whose primary function is to produce and secrete the Type 1 Collagen rich extracellular bone matrix. Sustained protein synthesis and matrix secretion is very demanding, both energetically and synthetically. How osteoblasts generate biomass, energy and other metabolites to sustain matrix production is not well understood. Cells metabolize diverse carbon sources to generate ATP, amino acids and antioxidants to support cellular activity. Glutamine is one such carbon source. In addition to direct incorporation in polypeptide chains, glutamine is an important oxidative fuel, a precursor of non‐essential amino acids, nucleotides and the anti‐oxidant glutathione (GSH). Thus, glutamine metabolism is likely a critical regulator of differentiation and bone formation in osteoblasts. Glutaminase (GLS) catalyzes the first and rate‐limiting step in glutamine metabolism, deaminating glutamine to form glutamate and ammonia. Previously, we demonstrated GLS activity is stimulated by Wnt signaling, a critical regulator of osteoblast differentiation. Importantly, GLS inhibition with Bis‐2‐(5‐phenylacetamido‐1,3,4‐thiadiazol‐2‐yl)ethyl sulfide (BPTES) significantly reduced bone mass in the Lrp5A214V/+ mouse model of human high bone mass disease. However, BPTES did not affect bone mass in wild type mice. Thus it is unclear if GLS is required for physiological bone formation or is simply overactive in pathological conditions. The objective of this study was to elucidate the role of GLS and glutamine metabolism during osteoblast differentiation. To this end, we employed a multifaceted approach to test if GLS is required for osteoblast differentiation. We used CRISPR/Cas9 technology to target Gls in primary calvarial osteoblasts (cOB). cOB lacking GLS are unable to differentiate into osteoblasts and display reduced GSH, increased reactive oxygen species (ROS) and diminished protein synthesis. Importantly, inhibiting GSH synthesis either pharmacologically or genetically using CRISPR phenocopied the effect of Gls deletion in cOB. Genetic deletion of a floxed Gls allele (Glsfl/fl) in mesenchymal progenitor cells using Prx1Cre significantly reduced bone mass in vivo. Mechanistically, this is due to a significant reduction in overall osteoblast numbers and diminished bone forming activity in differentiated osteoblasts. Reduced osteoblast numbers is due to aberrant specification of mesenchymal stem cells into the adipocyte rather than osteoblast lineage. Gls deletion in specified osteoblasts using Sp7Cre similarly reduced bone mass. Decreased bone mass in Sp7Cre;Glsfl/fl mice is the result of diminished osteoblast activity with no change in osteoblast specification or differentiation. Collectively, these data support a biphasic role for GLS during osteoblast differentiation. First, GLS functions in multipotent mesenchymal progenitors to promote osteoblast specification and differentiation; later, GLS acts in specified osteoblasts to promote robust bone forming activity. Our data suggest manipulation of GLS activity and glutamine metabolism may provide a valuable therapeutic approach for normalizing deranged osteoblast differentiation and bone anabolism associated with human bone diseases.