Osteoblastic γ-aminobutyric acid, type B receptors negatively regulate osteoblastogenesis toward disturbance of osteoclastogenesis mediated by receptor activator of nuclear factor κB ligand in mouse bone

The prevailing view is that signaling machineries for the neurotransmitterGABAare also expressed by cells outside the CNS. In cultured murine calvarial osteoblasts, mRNA was constitutively expressed for both subunits 1 and 2 of metabotropic GABA _B receptor (GABA _BR), along with inhibition by the GABA _BR agonist baclofen of cAMP formation, alkaline phosphatase (ALP) activity, and Ca ²⁺ accumulation. Moreover, baclofen significantly inhibited the transactivation of receptor activator of nuclear factor-κB ligand (RANKL) gene in a manner sensitive to a GABA _BR antagonist, in addition to decreasing mRNA expression of bone morphogenetic protein-2 (BMP2), osteocalcin, and osterix. In osteoblastic MC3T3-E1 cells stably transfected with GABA _BR1 subunit, significant reductions were seen in ALP activity and Ca ²⁺ accumulation, as well as mRNA expression of osteocalcin, osteopontin, and osterix. In cultured calvarial osteoblasts from GABA _BR1-null mice exhibiting low bone mineral density in tibia and femur, by contrast, both ALP activity and Ca ²⁺ accumulation were significantly increased together with promoted expression of both mRNA and proteins for BMP2 and osterix. No significant change was seen in the number of multinucleated cells stained for tartrate-resistant acid phosphatase during the culture of osteoclasts prepared from GABA _BR1-null mice, whereas a significant increase was seen in the number of tartrate-resistant acid phosphatase-positive multinucleated cells in co-culture of osteoclasts with osteoblasts isolated from GABA _BR1-null mice. These results suggest that GABA _BR is predominantly expressed by osteoblasts to negatively regulate osteoblastogenesis through down-regulation of BMP2 expression toward disturbance of osteoclastogenesis after down-regulation of RANKL expression in mouse bone.