Substance P stimulates late-stage rat osteoblastic bone formation through neurokinin-1 receptors.
Substance P (SP) is a widely distributed neuropeptide that works as a neurotransmitter and neuromodulator. Recently, SP receptors, particularly neurokinin-1 receptors (NK(1)-Rs) that have a high affinity for SP, have been observed not only in neuron and immune cells, but also in other peripheral cells, including bone cells. To identify the role of SP in bone formation, we investigated the expression of NK(1)-Rs in osteoblastic cells and the effects of SP on bone formation by rat calvarial osteoblastic cells. Rat calvarial osteoblastic cells were isolated and cultured for 3 weeks in alpha-MEM containing 10% serum, ascorbic acid, dexamethasone, and beta-glycerophosphate. We then investigated NK(1)-R expression, SP effects on osteoblastic bone formation, and osteocalcin mRNA expression in osteoblastic cells. RT-PCR and immunocytochemistry showed that NK(1)-R mRNA was expressed and NK(1)-R was present in 14-day, but not 7-day, cultured calvarial osteoblasts. Bone formation by cultured osteoblastic cells significantly increased after the addition of 10(-8)-10(-6)MSP. During 3 weeks of culture, the addition of SP in the first week did not significantly increase bone formation, whereas adding SP during the first and second week or all 3 weeks significantly increased calvarial osteoblastic bone formation. Furthermore, semi-quantitative RT-PCR indicated that SP stimulated osteocalcin mRNA expression in the osteoblasts at day 14 or day 21, whereas SP did not stimulated the runX2 or type I collagen mRNA expression at day 7 but stimulated them at day 14. These results indicate that SP stimulates bone formation by osteoblastic cells via NK(1)-Rs at late-stage bone formation. These effects were dependent on the expression of NK(1)-R in osteoblastic cells. Our findings suggest that SP secreted from sensory neurons may modulate bone formation after the expression of SP receptors.
Goto
T
T
Division of Anatomy, Kyushu Dental College, 2-6-1 Manazuru, Kitakyushu 803-8580, Japan. tgoto@kyu-dent.ac.jp
Nakao
K
K
Gunjigake
K K
KK
Kido
M A
MA
Kobayashi
S
S
Tanaka
T
T
D000818:Animals; D001846:Bone Development / Q000187:drug effects / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D003094:Collagen / Q000187:drug effects; D017931:DNA Primers; D004151:Dipeptides / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D018040:Receptors, Neurokinin-1 / Q000187:drug effects / Q000502:physiology*; D013373:Substance P / Q000031:analogs & derivatives / Q000494:pharmacology*
D017931:DNA Primers; D004151:Dipeptides; D007211:Indoles; D012333:RNA, Messenger; D018040:Receptors, Neurokinin-1; C078949:FK 888; D013373:Substance P; D003094:Collagen; C042867:spantide