Bone Abstracts

Introduction: Osteoblast lineage cells express glutamate receptors and secrete glutamate, which acts as an autocrine factor to promote cellular differentiation and activation. However, the mechanisms by which glutamate regulates these functions, remain unclear.

Methods: Primary osteoblasts were isolated from calvaria of 2–3 days old mice. The cells were treated with inhibitors of glutamate transporters, namely the Scl1a1 and Scl1a3 inhibitor L-serine-O-sulfate (SOS, 0.1 mM, 0.2 mM, 0.4 mM) and the Scl1a1-4 inhibitor DL-threo-hydroxy-aspartate (THA, 0.25 mM, 0.5 mM, 1.0 mM) for 3 and 5 days. 1.25(OH)₂D₃ (10 nM) and TNF-α (5 ng/ml) were also used. Gene expression was analyzed by real-time PCR. ALP activity and the number of viable cells were assessed. Glutamate concentrations were determined by the glutamate oxidase method.

Results: Transcripts encoding glutamate transporters (Scl1a1-5) and glutamate receptors (APAM3, Grina, Grinal1a and NMDA2D) were expressed in primary mouse osteoblasts. Expression levels of Scl1a1, Scl1a3 and Grina were upregulated by 1.25(OH)₂D₃ and TNF-α. Inhibition of Scl1a by SOS and THA led to an increase in the concentration of extracellular glutamate in a dose and time dependent manner. SOS and THA decreased osteoblast proliferation, but stimulated osteoblast differentiation in a dose dependent manner. The expression of osteocalcin and type I collagen, two markers of osteoblast differentiation was also upregulated upon inhibition of glutamate transporters. Scl1a block by SOS and THA acts synergistically with 1.25(OH)₂D₃ to stimulate osteoblast differentiation.

Conclusion: Our study demonstrates that glutamate transport is involved in osteoblast differentiation. Inhibition of Slc1a promotes osteoblast differentiation by increasing the concentration of extracellular glutamate. Slc1a transporters control the extracellular glutamate concentrations, and by this mechanism contribute to the stimulation of osteoblast differentiation and activation. This suggests that members of the Slc1a family of glutamate transporters can serve as potential therapeutic targets to modulate the differentiation of osteoblast lineage cells.