Fig 2: Osteoblast-derived ProCT inhibits early osteoclastogenesis. a Representative TRAP staining images of WT osteoclasts on Day 4 of differentiation, after treatment with osteoblast conditioned medium (CM) supplemented with M-CSF, RANKL and/or ProCT (200 ng·mL-1) for 24 h (scale bar = 100 µm). CM was collected from calvarial osteoblasts (Day 10 of differentiation) derived from mice of the indicated genotypes and stimulated with iPTH for 24 h. The quantification of osteoclast number per field of view is shown on the left (N.Ocl). n = 4 independent cultures per group. b Quantification of N.Ocl in the murine monocytic cell line RAW 264.7 or human peripheral blood mononuclear cells (hPBMCs) on Day 5 of osteoclast differentiation. n = 4 independent cultures per group. c Quantification of the number of TRAP-positive, multinucleated cells (N.Ocl) in murine bone marrow-derived osteoclast cultures continuously stimulated with ProCT (200 ng·mL-1) beginning on the indicated days of differentiation. n = 3-4 independent cultures per group. d Gene expression levels (virtual copy numbers) of the osteoclast marker Calcr as a control (left) and the indicated CGRP receptor components (Calcrl and Ramp1) in murine bone marrow-derived osteoclasts during the course of differentiation. n = 3 independent cultures per group. In (a–d), one-way ANOVA followed by Tukey’s post hoc test was applied. e Immunofluorescence staining using a CLR-specific antibody in trabecular bone (spine sections) of untreated WT mice

Fig 3: The impact of mechanical loading and neuropeptide stimulation on sensory neuropeptide receptor gene expression of RAW264.7 cells. (A) Relative gene expression of NK1R, CRLR and Ramp1 after 4 h loading per day on two consecutive days in relation to unloaded cells (= calibrator, RQ = 1) was analyzed using quantitative real-time PCR. Normalizer: GAPDH. One sample t-test ** p < 0.01; *** p < 0.001. NK1R, CRLR n = 20, Ramp1 n = 5; (B–D) Receptor gene expression was determined after 2 days of loading for 4 h per day in the presence of SP (B, for NK1R) or aCGRP (C for CRLR, D for Ramp1) using quantitative real-time PCR. Normalizer: GAPDH. Gene expression of stimulated cells was calibrated to the expression of unstimulated cells (= calibrator, RQ = 1). One sample t-test * p < 0.05; ** p < 0.01; *** p < 0.001. n = 5. NK1R—neurokinin receptor 1, CRLR—calcitonin receptor-like receptor, Ramp1—receptor activity modifying protein 1, RQ—relative quantification, SP—substance P, PCR—polymerase chain reaction, GAPDH—glyceraldehyde 3-phosphate dehydrogenase, aCGRP—alpha-calcitonin gene-related peptide.

Fig 4: The impact of mechanical loading on sensory neuropeptide receptor protein expression of RAW264.7 cells. Receptor protein expression of NK1R (A), CRLR (B) and Ramp1 (C) was analyzed using the Western Blotting of lysates from cells loaded for 4 h/day on 1, 2 and 3 consecutive days. Expression of ß-actin served as endogenous loading control (=100% line). Mann–Whitney test * p < 0.05; ** p < 0.01; *** p < 0.001. n = 7–8; (D) Representative Western Blot pictures for the CRLR (~53 kDa), NK1R (~46 kDa), Ramp1 (~17 kDa) and ß-actin (~37 kDa, endogenous control) of control cells and cells loaded for 1, 2 and 3 consecutive days (presenting 2 lanes for each condition). NK1R—neurokinin receptor 1, CRLR—calcitonin receptor-like receptor, Ramp1—receptor activity modifying protein 1.

Fig 5: CGRP and CGRP receptor are expressed in the fracture callus on mRNA level. (a) Relative serum CGRP levels in WT and aCGRP-deficient mice with a femoral osteotomy stabilized with an external fixator 14 days post-injury. n = 4–6 mice per group (unpaired student's t-test). (b, c) Gene expression (virtual copy numbers per Gapdh) of the indicated genes in the intact femoral shaft of control animals (intact) and in the fracture callus at the respective time points during bone regeneration (d3, d7, d14). Calca = encoding aCGRP; Crlr = calcitonin receptor-like receptor; Ramp1 = receptor activity-modifying protein 1. n = 4–6 mice per group (one-way Anova followed by Tukey post-hoc test). (d) Calca expression (fold) in the indicated tissues 3 and 7 days following Fx. Hypoth. = hypothalamus; WAT = white adipose tissue; BAT = brown adipose tissue; n.d. = not detectable. The dotted red line indicates average expression of respective, untreated control tissues. n = 3–5 mice per group (unpaired student's t-test). For (a-d), box plots represent median with minimum and maximum whiskers. Controls are untreated mice of the same sex and age.