Substance P stimulates bone marrow stromal cell osteogenic activity, osteoclast differentiation, and resorption activity in vitro

Abstract

Introduction: 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.

Materials and 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.

Fig. 1

Laser scanning confocal microscopy of mouse bone marrow stromal cells (BMSCs). Alkaline phosphatase and the substance P (SP) NK1 receptors were co-localized in the cytoplasm and on the plasma membrane of BMSCs. (A) The punctuated presence of green fluorescence indicates the presence of alkaline phosphatase. (B) The homogeneous presence of red fluorescence indicates the presence of NK1 receptors. (C) The presence of yellow fluorescence indicates the presence of the two proteins overlapping in the cellular microcompartment. (D) RT-PCR analysis of NK1 receptor gene (TACR-1) expression in mouse BMSCs demonstrates stable levels over the cell life cycle. The mRNA gel band of TACR-1 and 18S at respective time points were quantified using densitometry, separately. (E) NK1 mRNA is present in BMSCs, murine osteoblast-like MC3T3-E1 cells, and mouse brain.

Fig. 2

Effects of SP treatment (10⁻⁸ M, 10⁻¹⁰ M, 10⁻¹² M) on BMSC cellular proliferation. (A) The highest concentration of SP (10⁻⁸ M) did increase BrdU incorporation in mouse BMSCs at 72 h post-seeding. Relative changes were calculated by the percentage of cells positive for BrdU incorporation relative to total nuclei positive for propidium iodide. SP treatment for 21 days had no effect in the later stages of BMSC cellular proliferation as measured by (B) crystal violet staining or (C) soluble cellular protein at 21 days post-seeding. Values are means ± SEM for six wells (* p <0.05 vs. control).

Fig. 3

This figure illustrates the time course of BMSC differentiation in vitro. BMSC osteoblastic gene expression was evaluated on days 3, 7, 14, and 21 post-seeding, as measured by real time PCR for (A) alkaline phosphatase, (B) osteocalcin, (C) collagen type I, and (D) Runx2 mRNA levels. Gene expression increased for all osteoblastic markers over time. Values are means ± SEM for six independent cultures (** p <0.01; *** p <0.001 vs. day 3 mRNA levels).

Fig. 4

Effects of 3, 7, 14, and 21 days of SP treatment (10⁻⁸ M, 10⁻¹⁰ M, 10⁻¹² M) on mouse BMSC gene expression measured by real time PCR for (A) alkaline phosphatase, (B) osteocalcin, (C) Runx2, and (D) collagen type I mRNA levels (* p <0.05 vs. the day 3 mRNA level for the same SP concentration). Effects of SP on osteoblast-specific protein expression in mouse BMSCs were also measured on days 7, 14, and 21 post-seeding. (E) Alkaline phosphatase activity was determined by staining with a solution consisting of equal parts p-nitrophenol phosphate (Sigma) and alkaline buffer solution (Sigma) and then was normalized by cell number determined by crystal violet staining. Values are means ± SEM for six wells (* p <0.05 vs. control). Levels of (F) osteocalcin in culture medium and (G) Runx2 produced by BMSCs were determined by ELIA and western blot, respectively. (H) Effect of 21 days of SP treatment on mineralization in BMSCs was determined by Alizarin red staining and normalized to crystal violet staining. Values are means ± SEM for six wells (** p <0.01 vs. control).

Fig. 5

Laser scanning confocal microscopy of bone marrow macrophages (BMMs) and osteoclasts derived from M-CSF and RANKL stimulated mouse non-adherent bone marrow cells. (A) Green immunofluorescence indicates the presence of tartrate-resistant acid phosphatase (TRAP⁺), a marker of osteoclasts and osteoclast precursors. (B) Red indicates the presence of NK1 receptors. (C) Yellow indicates the presence of the two proteins in the same cellular microcompartment. (D) Green immunofluorescence indicates the presence of CD 14, a marker of monocyte/macrophages. (E) Red indicates the presence of NK1 receptors. (F) The presence of yellow fluorescence indicates the presence of the two proteins in the same cellular microcompartment. (G) RT-PCR measurements of NK1 receptor mRNA levels in mouse BMMs demonstrate a slight decline in NK1 expression at day 7. The mRNA gel band densities of TACR-1 and 18S at respective time points were quantified using densitometry, separately. (H) NK1 receptor mRNA is present in BMMs, RAW 264.7 cells, and mouse brain. Values are means ± SEM for six wells (*** p <0.001 vs. day 4).

Fig. 6

Effects of SP treatment on BMM (A) BrdU incorporation, (B) pit erosion area, and (C) multinucleated TRAP ⁺ cell numbers in BMMs. (D) Effect of SP treatment on multinucleated TRAP ⁺ cell numbers in RAW 264.7 cell culture. SP (10⁻⁸M) treatment had no effect on BrdU incorporation, but dramatically increased the number of TRAP⁺ cells and the pit erosion area, as compared to controls. (E) Photomicrographs illustrating the effects of SP (10⁻⁸ M) on TRAP⁺ cell numbers and dentine disc erosion area in BMMs (** p <0.01; *** p <0.001 vs. control).

Fig. 7

Effects of SP on NF-κB nuclear translocation in BMMs and RANKL production in BMSCs. (A) RANKL (100 ng/ml) induced NF-κB nuclear translocation in BMMs in a time dependent manner. (B) SP (10⁻⁸ M) also induced NF-κB nuclear translocation in BMMs in RANKL free media and there was no additive effect with RANKL. (C) SP concentration-dependently stimulated RANKL protein levels in the cell layer of BMSC cultures (* p <0.05 vs. control).