Evidence for targeted vesicular glutamate exocytosis in osteoblasts

Regulated intercellular signaling is essential for the maintenance of bone mass. In recent work we described how osteoblasts and osteoclasts express f unctional receptors for the excitatory amino acid, glutamate, indicating th at a signaling pathway analogous to synaptic neurotransmission exists hi bo ne. Here, we show that osteoblasts also express the essential molecular fra mework for regulated glutamate exocytosis to occur as is present in presyna ptic neurons. A combination of reverse transcription-polymerase chain react ion (RT-PCR) and northern and western blotting is used to show expression o f the target membrane-SNARE (soluble NSF attachment protein receptor), prot eins SNAP-25 and syntaxin 4 and the vesicular-SNARE protein VAMP (synaptobr evin), the minimum molecular requirements for core exocytotic complex forma tion. Immunofluorescent localizations reveal peripheral SNAP-25 expression on osteoblastic cells, particularly at intercellular contact sites, colocal izing with immunoreactive glutamate and the synaptic vesicle-specific prote in, synapsin I. We also identify multiple accessory proteins associated wit h vesicle trafficking, including munc18, rSec8, DOC2, syntaxin 6, and synap tophysin, which have varied roles in regulated glutamate exocytosis. mRNA f or the putative Ca2+-dependent regulators of vesicle recycling activity, sy naptotagmin I (specialized for fast Ca2+- dependent exocytosis as seen in s ynaptic neurotransmission), and the GTP-binding protein Rab3A are also iden tified by northern blot analysis. Finally, we demonstrate that osteoblastic cells actively release glutamate in a differentiation-dependent manner. Th ese data provide compelling evidence that osteoblasts are able to direct gl utamate release by regulated vesicular exocytosis, mimicking presynaptic gl utamatergic neurons, showing that a process with striking similarity to syn aptic neurotransmission occurs in bone. (C) 2001 by Elsevier Science Inc. A ll rights reserved.