Fig 2: DRG‐derived CGRP promotes CHSY1 expression in NP cell via RAMP1/CREB signaling. A) Quantitative RT‐PCR analysis of CHSY1 expression from NP cells treated with Vehicle, VIP, CGRP, NPY, and SP at the concentration of 10–7 m, respectively. B,C) Representative images of WB and quantitative analysis of CHSY1 expression for NP cells treated with Vehicle, VIP, CGRP, NPY, and SP. D) Quantitative analysis of CS by DMMB assay for NP cells treated with Vehicle, VIP, CGRP, NPY, and SP. E,F) Representative immunofluorescence staining of CS‐56 (green) and quantitative analysis for NP cells treated with Vehicle, VIP, CGRP, NPY, and SP. Scale bar: 50 µm. G) Schematic graph for in vitro co‐culture system of DRG sensory neuros and NP cells treated with CGRP neutralizing antibody. H) Quantitative RT‐PCR analysis of CHSY1 expression from NP cells in the DRG co‐culture system treated with CGRP neutralizing antibody and vehicle. I,J) Representative images of WB and quantitative analysis of CHSY1 expression for NP cells in the DRG co‐culture system treated with CGRP neutralizing antibody and vehicle. K) Quantitative analysis of CS by DMMB assay for NP cells in the DRG co‐culture system treated with CGRP neutralizing antibody and vehicle. L,M) Representative immunofluorescence staining of CS‐56 (green) and quantitative analysis for NP cells in the DRG co‐culture system treated with CGRP neutralizing antibody and vehicle. Scale bar: 50 µm. N,O) Representative images of WB and quantitative analysis of pCREB expression for NP cells treated with CGRP for 0–120 min respectively. P,Q) Representative images of WB and quantitative analysis of CHSY1 expression for NP cells treated with CGRP, si‐RAMP1, CGRP+si‐RAMP1, CREB inhibitor 666‐15, and CGRP+666‐15, respectively. R) Quantitative analysis of CS by DMMB assay for NP cells treated with CGRP, si‐RAMP1, CGRP+si‐RAMP1, CREB inhibitor 666‐15, and CGRP + 666‐15, respectively. All data are presented as means ± SEM, n = 6 per group, *P < 0.05 and **P < 0.01 (A,C,D,F,O,Q,R). Statistical significance was determined by two‐tailed Student's t‐test. *P < 0.05 and **P < 0.01. Statistical significance was determined by one‐way ANOVA (H,J,K,M).

Fig 3: Investigation of periodontal tissue destruction and healing in wild-type and Ramp1−/− mice using micro computed tomography (μCT) imaging. (a) μCT images of wild-type and Ramp1−/− mice on ligation days 0, 3, 7, 10, and 14. (b) As indicated by the yellow line, the distance from the cemento-enamel junction to the alveolar crest was measured for four roots: the distal root of the maxillary first molar, the mesial and distal roots of the maxillary second molar, and the mesial root of the maxillary third molar, and the total was evaluated as the Total Bone Loss. (c) Total Bone Loss of wild-type and Ramp1−/− mice at days 0–14 is shown in the graph Statistical significance was determined by Tukey’s test (c) (blue: wild-type mice, red: Ramp1−/− mice, n = 5, *P < 0.05, **P < 0.01, ***P < 0.001). No significant differences in alveolar bone status between the wild-type and Ramp1−/− mice were observed on day 0. Ramp1−/− mice showed delayed alveolar bone recovery after silk thread removal compared with that in wild-type mice.

Fig 4: Expression of calcitonin gene-related peptide (CGRP) receptor in mouse periodontal tissue and MPDL22 cells. (a) Receptor activating modifying protein 1 (Ramp1), calcitonin receptor-like receptor (Clr), and receptor component protein (Rcp) expression in MPDL22 cells was analyzed using reverse transcription-polymerase chain reaction. Gene expression was observed in all the components. (b) A whole cell fraction of MPDL22 cells was collected and subjected to western blotting with a specific antibody for RAMP1. RAMP1-positive reaction was observed. Original blots/gels are presented in Supplementary Fig. 1 (a, b1, b2). (c) Immunostaining image of a mouse maxillary first molar without primary antibody (control). (d) Immunostaining image of a mouse maxillary first molar with anti-RAMP1 antibody. (e) Enlarged image of the upper left square in D. RAMP1-positive reaction was observed in the entire gingiva except the stratum corneum. (f) Enlarged image of the lower right square of D. RAMP1-positive reaction was observed in the pulp and periodontal ligament. AB alveolar bone, D dentin, G gingiva, P pulp, PDL periodontal ligament. Scale bar: 500 μm (c,d) 100 μm (e,f).

Fig 5: Effect of calcitonin gene-related peptide (CGRP) on the process of induction of cytodifferentiation of MPDL22 cells into hard tissue-forming cells. (a) Real-time polymerase chain reaction of Ramp1 and Clr during the cytodifferentiation process in the absence of CGRP. On day 3, the expression of CGRP receptor was significantly increased. (b) Induced cytodifferentiation in the presence of 10−14 M, 10−13 M, 10−12 M, 10−11 M, and 10−10 M CGRP, Real-time PCR was performed for Osterix on day 3, Alp on day 6, and Osteocalcin on day 12. CGRP stimulation caused calcification in MPDL22 cells. CGRP stimulation significantly upregulated the expression of calcification-related genes (Osterix, Alp, and Osteocalcin) in MPDL22 cells. (c) Alizarin staining image on day 18 after induction of cytodifferentiation CGRP stimulation significantly promoted the formation of calcified nodules. Statistical significance was determined by Tukey’s test (a,b) and Student's-t test (c). *P < 0.05, **P < 0.01, ***P < 0.001.