Calcitonin-gene-related peptide stimulates stromal cell osteogenic differentiation and inhibits RANKL induced NF-kappaB activation, osteoclastogenesis and bone resorption - PubMed (original) (raw)

Liping Wang et al. Bone. 2010 May.

Abstract

Previously we observed that capsaicin treatment in rats inhibited sensory neuropeptide signaling, with a concurrent reduction in trabecular bone formation and bone volume, and an increase in osteoclast numbers and bone resorption. Calcitonin-gene-related peptide (CGRP) is a neuropeptide richly distributed in sensory neurons innervating the skeleton and we postulated that CGRP signaling regulates bone integrity. In this study we examined CGRP effects on stromal and bone cell differentiation and activity in vitro. CGRP receptors were detected by immunocytochemical staining and real time PCR assays in mouse bone marrow stromal cells (BMSCs) and bone marrow macrophages (BMMs). CGRP effects on BMSC proliferation and osteoblastic differentiation were studied using BrdU incorporation, PCR products, alkaline phosphatase (ALP) activity, and mineralization assays. CGRP effects on BMM osteoclastic differentiation and activity were determined by quantifying tartrate-resistant acid phosphatase positive (TRAP(+)) multinucleated cells, pit erosion area, mRNA levels of TRAP and cathepsin K, and nuclear factor-kappaB (NF-kappaB) nuclear localization. BMSCs, osteoblasts, BMMs, and osteoclasts all expressed CGRP receptors. CGRP (10(-10)-10(-8) M) stimulated BMSC proliferation, up-regulated the expression of osteoblastic genes, and increased ALP activity and mineralization in the BMSCs. In BMM cultures CGRP (10(-8) M) inhibited receptor activator of NF-kappaB ligand (RANKL) activation of NF-kappaB. CGRP also down-regulated osteoclastic genes like TRAP and cathepsin K, decreased the numbers of TRAP(+) cells, and inhibited bone resorption activity in RANKL stimulated BMMs. These results suggest that CGRP signaling maintains bone mass both by directly stimulating stromal cell osteoblastic differentiation and by inhibiting RANKL induced NF-kappaB activation, osteoclastogenesis, and bone resorption.

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Conflict of interest statement

Conflict of Interest: None of the authors have financial interests that might be construed as affecting the conduct or reporting of this work.

Figures

Fig. 1

Fig. 1

Laser scanning confocal microscopy demonstrated the presence of the calcitonin gene-related protein (CGRP) receptors in adherent mouse bone marrow stromal cells (BMSCs). The CGRP receptor is a dimer complex of two molecules, the calcitonin receptor-like receptor (CRL) and a receptor activity-modifying protein (RAMP1), both of which are required for physiologic activation by CGRP. Alkaline phosphatase and the CRL receptor co-localized in the cytoplasm and cell membrane of BMSCs on day 14 of cell culture. The presence of green fluorescence indicates the presence of alkaline phosphatase (A), the presence of red fluorescence indicates the presence of CRL (B), and the presence of yellow fluorescence indicates co-labeling for the two proteins in the same cellular microcompartment (C). RAMP1 and CRL co-localized in the BMSC derived osteoblasts on day 14 (D, E, and F) and in MC3T3-E1 osteoblast-like cells (G, H, and I) on day 7 of cell culture. RAMP 1 immunofluorescence (green) was observed in the cytoplasm, cell membrane, and nucleus (D, G) and CRL immunostaining (red) was localized in the cytoplasm and on the plasma membrane (E, H). The presence of yellow fluorescence indicates co-labeling for the two proteins in the same cellular microcompartment (F, I). Real time PCR analysis of RAMP1 (J) and CLR (K) mRNA levels are shown for mouse BMSCs, brain and MC3T3-E1 cells. RAMP1 mRNA levels in BMSC cultures gradually dropped from day 3 to day 21 post-seeding, while CRL expression remained stable over time. Values are means ± SEM for six wells and CRL and RAMP1 mRNA levels were normalized to 18S mRNA levels.

Fig. 2

Fig. 2

Effects of 7, 14, and 21 days of CGRP treatment (10-10 M and 10-8 M) on mouse BMSC gene expression measured by real time PCR for (A) alkaline phosphatase, (B) collagen type I, (C) osteocalcin, and (D) Runx2 mRNA levels. CGRP stimulated osteoblastic gene expression in the earlier stages of osteoblastic cell differentiation. Values are means ± SEM for six wells. * p <0.05, ** p <0.01, *** p <0.001 vs. control.

Fig. 3

Fig. 3

Panel A illustrates the effects of 14 and 21 days of CGRP (10-14 M, 10-12 M, 10-10 M, 10-8 M) treatment on alkaline phosphatase activity in mouse adherent BMSCs. Cell lysates were used for analyzing alkaline phosphatase activity and activity was normalized to the amount of cell protein. The 10-10 M concentration of CGRP significantly stimulated alkaline phosphatase activity in BMSCs at both time points. CGRP (10-12 - 10-8 M) added to the BMSC culture medium for 21 days increased mineralization in BMSCs as determined by Alizarin red staining (B) and reduced cell proliferation, as estimated by crystal violet staining (C). When mineralization was normalized to cell number (D) there was a 923% increase after treatment with CGRP (10-10 M) and a 614% increase after treatment with CGRP (10-8 M). Values are means ± SEM for six wells. * p<0.05, ** p< 0.01, *** p<0.001 vs. control.

Fig. 4

Fig. 4

Confocal microscopy demonstrating CGRP receptors in M-CSF and RANKL stimulated mouse nonadherent bone marrow stromal cell cultures at day 7 post-seeding. Under these conditions the majority of stromal cells in the culture medium are transformed into bone marrow macrophages (BMMs), preosteoclasts, and osteoclasts. Green fluorescence indicates the presence of tartrate-resistant acid phosphatase (TRAP), a marker for osteoclasts and osteoclast precursors (A), red fluorescence indicates CRL receptors (B), and yellow fluorescence indicates the co-labeling of the two proteins in the same cellular microcompartment (C). RAMP 1 and CLR immunostaining was observed in monocytes and multinucleated osteoclasts in BMM cultures at 7 days post-seeding. RAMP1 immunofluorescence (green) was observed in the cytoplasm, cell membrane, and nucleus (D) and CRL immunostaining (red) was localized in the cytoplasm and plasma membrane (E). Yellow fluorescence indicates co-staining for the two proteins in the same cellular microcompartment (F). Real time PCR measurements of RAMP1 (G) and CRL (H) mRNA levels are shown in BMM cultures at days 4 and 7 post-seeding. Values are means ± SEM for six wells and CRL and RAMP1 mRNA levels were normalized to 18S mRNA levels. *** p <0.001 vs. day 4.

Fig. 5

Fig. 5

Effects of CGRP on osteoclast formation and resorption in M-CSF and RANKL stimulated mouse nonadherent bone marrow stromal cell cultures. Osteoclast formation was measured by counting multinucleated TRAP+ cells per well (A) and osteoclast resorption was evaluated by measuring total erosion area on BD BioCoat osteologic disc (B). CGRP (10-8 M) treatment reduced osteoclastogenesis and bone resorption in the BMM cell cultures. Values for TRAP+ cells are means ± SEM for six wells, and erosive areas are means ± SEM for six to eight discs. Panel C shows representative photomicrographs of TRAP+ osteoclasts in controls (a) and after CGRP (10-8 M) treatment (b) and the erosion area in controls (c) and after CGRP (10-8 M) treatment (d). * p <0.05, ** p <0.01 vs. control.

Fig. 6

Fig. 6

Effects of 7 days of CGRP treatment on TRAP (A) and cathepsin K (B) gene expression in M-CSF and RANKL stimulated nonadherent bone marrow stromal cell cultures. CGRP (10-8 M) reduced TRAP mRNA levels by 33% and cathepsin K levels by 23% compared to controls not treated with CGRP. Panel C shows the effects of RANKL (100ng/mL), CGRP (10-8 M), and the combination of RANKL (100ng/mL) and CGRP (10-8 M) on nuclear NF-κB-p65 levels in BMMs after 30 minutes of treatment. RANKL activated NF-κB in BMMs and CGRP completely blocked this effect. CGRP alone had no effect on NF-κB activation. The densitometry reading of each band was normalized by its β actin value. The readings shown at the bottom of the panel are the relative fold change over the values of control BMM samples treated only with M-CSF. * p <0.05, ** p <0.01 vs. control.

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