title: Substance P stimulates bone marrow stromal cell osteogenic activity, osteoclast differentiation, and resorption activity in vitro.
abstract: BACKGROUND
  SP is a neuropeptide distributed in the sensory nerve fibers that innervate the medullar tissues of bone, as well as the periosteum. Previously we demonstrated that inhibition of neuropeptide signaling after capsaicin treatment resulted in a loss of bone mass and we hypothesized that SP contributes to bone integrity by stimulating osteogenesis.
  
  
  METHODS
  Osteoblast precursors (bone marrow stromal cells, BMSCs) and osteoclast precursors (bone marrow macrophages, BMMs) derived from C57BL/6 mice were cultured. Expression of the SP receptor (NK1) was detected by using immunocytochemical staining and PCR. Effects of SP on proliferation and differentiation of BMSCs were studied by measuring BrdU incorporation, gene expression, alkaline phosphatase activity, and osteocalcin and Runx2 protein levels with EIA and western blot assays, respectively. Effects of SP on BMMs were determined using a BrdU assay, counting multinucleated cells staining positive for tartrate-resistant acid phosphatase (TRAP(+)), measuring pit erosion area, and evaluating RANKL protein production and NF-kappaB activity with ELISA and western blot.
  
  
  RESULTS
  The NK1 receptor was expressed in both BMSCs and BMMs. SP stimulated the proliferation of BMSCs in a concentration-dependent manner. Low concentrations (10(-12) M) of SP stimulated alkaline phosphatase and osteocalcin expression, increased alkaline phosphatase activity, and up-regulated Runx2 protein levels, and higher concentrations of SP (10(-8) M) enhanced mineralization in differentiated BMSCs. SP also stimulated BMSCs to produce RANKL, but at concentrations too low to evoke osteoclastogenesis in co-culture with macrophages in the presence of SP. SP also activated NF-kappaB in BMMs and directly facilitate RANKL-induced macrophage osteoclastogenesis and bone resorption activity.
  
  
  CONCLUSIONS
  NK1 receptors are expressed by osteoblast and osteoclast precursors and SP stimulates osteoblast and osteoclast differentiation and function in vitro. SP neurotransmitter release from sensory neurons could potentially regulate local bone turnover in vivo.
authors.affiliation: Physical Medicine and Rehabilitation Service (117), Veterans Affairs Palo Alto Health Care System, 3801 Miranda Ave., Palo Alto, CA 94304, USA. lipingw@stanford.edu
authors.forename: Liping
authors.initials: L
authors.lastname: Wang
authors.forename: Rong
authors.initials: R
authors.lastname: Zhao
authors.forename: Xiaoyou
authors.initials: X
authors.lastname: Shi
authors.forename: Tzuping
authors.initials: T
authors.lastname: Wei
authors.forename: Bernard P
authors.initials: BP
authors.lastname: Halloran
authors.forename: David J
authors.initials: DJ
authors.lastname: Clark
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Jacobs
authors.forename: Wade S
authors.initials: WS
authors.lastname: Kingery
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D001862:Bone Resorption / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D051379:Mice; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects; D053245:RANK Ligand / Q000378:metabolism / Q000494:pharmacology; D011971:Receptors, Immunologic / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013373:Substance P / Q000494:pharmacology*
chemical_list: D016328:NF-kappa B; D053245:RANK Ligand; D011971:Receptors, Immunologic; D013373:Substance P; D016173:Macrophage Colony-Stimulating Factor