Abstract
Mechanical loading is a potent stimulator of bone formation. A screen for genes associated with mechanically-induced osteogenesis implicated the glutamate transporter GLAST-1 (1), in the mechanoresponse. We are investigating whether modulation of glutamate transporters represents a potential anabolic therapy in bone. Bone cells express functional components from each stage of the glutamate signalling pathway and activation of ionotropic glutamate receptors on osteoblasts can increase bone forming activity (2). Five high affinity Na+-dependant excitatory amino acid transporters (EAATs 1-5) regulate glutamatergic signalling. EAAT1 (GLAST-1) is expressed by osteocytes and bone-forming osteoblasts in vivo.
We quantified transcripts for EAATs 1-3 and two splice variants (EAAT1a and EAAT1ex9skip) in human osteoblasts (MG63, SaOS-2 and primary) using real time-PCR. EAAT1a expression was very low whilst levels of the dominant negative EAAT1ex9skip were much higher in all cell types. EAAT1 and EAAT3 proteins were detected by immunofluorescence. We also demonstrated that glutamate transporters function in human osteoblasts. Sodium-dependent 14C-labelled glutamate uptake, sensitive to pharmacological EAAT inhibitors (t-PDC, TBOA) and extracellular glutamate concentration (10-500μM) was detected in MG63 and SaOS-2 cells.
To determine whether modulation of EAATs can influence bone formation, we used pharmacological inhibitors of EAATs 1-5 (t-PDC and TBOA) and also over-expressed EAAT1exon9skip using antisense oligonucleotides (AONs) targeted to splice donor sequence of exon 9. Experiments were performed in 0-500μM glutamate. Pharmacological inhibition of EAATs over 5-21 days increased alkaline phosphatase activity and mineralisation of SaOS-2 cells and human primary osteoblasts. Over-expression of EAAT1ex9skip significantly increased cell number and decreased cell death as well as significantly increasing PCNA, Osteonectin and Type I collagen mRNAs in MG63 cells. Furthermore, over-expression of EAAT1ex9skip increased mean alkaline phosphatase activity over 48hrs in SaOS-2 cells.
These data show that EAATs are expressed and functional in osteoblasts and that pharmaceutical and genetic inhibition of their activity increases bone formation. These mechanically regulated glutamate transporters are important in regulating bone homeostasis and their manipulation may represent a new anabolic therapy for the treatment of disorders such as osteoporosis or non-union fractures.
