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. 2024 Jun 2;19(1):330.
doi: 10.1186/s13018-024-04805-w.

Involvement of RAMP1/p38MAPK signaling pathway in osteoblast differentiation in response to mechanical stimulation: a preliminary study

Thunwa Binlateh  1 , Chidchanok Leethanakul  2 , Peungchaleoy Thammanichanon  3   4
Affiliations

Involvement of RAMP1/p38MAPK signaling pathway in osteoblast differentiation in response to mechanical stimulation: a preliminary study

Thunwa Binlateh et al. J Orthop Surg Res. .

Abstract

Objective: The present study aimed to investigate the underlying mechanism of mechanical stimulation in regulating osteogenic differentiation.

Materials and methods: Osteoblasts were exposed to compressive force (0-4 g/cm2) for 1-3 days or CGRP for 1 or 3 days. Expression of receptor activity modifying protein 1 (RAMP1), the transcription factor RUNX2, osteocalcin, p38 and p-p38 were analyzed by western blotting. Calcium mineralization was analyzed by alizarin red straining.

Results: Using compressive force treatments, low magnitudes (1 and 2 g/cm2) of compressive force for 24 h promoted osteoblast differentiation and mineral deposition whereas higher magnitudes (3 and 4 g/cm2) did not produce osteogenic effect. Through western blot assay, we observed that the receptor activity-modifying protein 1 (RAMP1) expression was upregulated, and p38 mitogen-activated protein kinase (MAPK) was phosphorylated during low magnitudes compressive force-promoted osteoblast differentiation. Further investigation of a calcitonin gene-related peptide (CGRP) peptide incubation, a ligand for RAMP1, showed that CGRP at concentration of 25 and 50 ng/ml could increase expression levels of RUNX2 and osteocalcin, and percentage of mineralization, suggesting its osteogenic potential. In addition, with the same conditions, CGRP also significantly upregulated RAMP1 and phosphorylated p38 expression levels. Also, the combination of compressive forces (1 and 2 g/cm2) with 50 ng/ml CGRP trended to increase RAMP1 expression, p38 activity, and osteogenic marker RUNX2 levels, as well as percentage of mineralization compared to compressive force alone. This suggest that RAMP1 possibly acts as an upstream regulator of p38 signaling during osteogenic differentiation.

Conclusion: These findings suggest that CGRP-RAMP1/p38MAPK signaling implicates in osteoblast differentiation in response to optimal magnitude of compressive force. This study helps to define the underlying mechanism of compressive stimulation and may also enhance the application of compressive stimulation or CGRP peptide as an alternative approach for accelerating tooth movement in orthodontic treatment.

Keywords: CGRP; Mechanical loading; Osteoblast; RAMP1.

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Conflict of interest statement

The authors have no conflicts of interests to declare.

Figures

Fig. 1
Fig. 1
A A diagram presenting isolation of primary alveolar osteoblast cells. B Compressive force application model, performed in a 24-well plate
Fig. 2
Fig. 2
Viability of cells analyzed by MTT assay after being treated with 1–4 g/cm2 for 1–3 days. Data are expressed as mean ± SD (n = 5 for each group in triplicate); *p < 0.05 and **p < 0.01 compared with the control group, one-way ANOVA
Fig. 3
Fig. 3
Effect of different magnitudes and durations of compressive force on osteoblast differentiation. (A, C, E) Alizarin red staining, and (B, D, F) Percentage of mineralization relative to control cells after being treated with 1–4 g/cm2 for 1–3 days on day 7, 14 and 21. Data are expressed as mean ± SD (n = 5 for each group in triplicate); *p < 0.05 and **p < 0.01 compared with the control group, one-way ANOVA
Fig. 4
Fig. 4
Effect of compressive forces on osteogenic marker expression. (A) Immunoblotting analysis of RUNX2 and osteocalcin, (B) Quantitative expression levels of RUNX2 and osteocalcin after being treated with 1 and 2 g/cm2 for 1 day. Data are expressed as mean ± SD; (n = 5 for each group in triplicate); *p < 0.05 and **p < 0.01 compared with the control group, one-way ANOVA
Fig. 5
Fig. 5
Compressive force upregulates RAMP1 and activates p38. (A) Immunoblotting analysis of RAMP1 p38, and p-p38, (B) Quantitative expression levels of RAMP1 p38, and p-p38, and (C) Expression ratio of p-p38/p38 after being treated with 1 and 2 g/cm2 for 1 day. Data are expressed as mean ± SD(n = 5 for each group in triplicate); *p < 0.05 and **p < 0.01 compared with the control group, one-way ANOVA
Fig. 6
Fig. 6
CGRP peptide promotes osteoblast differentiation. (A) Viability of cells after being incubated with CGRP peptide at concentrations of 25–200 ng/ml for 1–3 days. (B) Alizarin red staining, and (C) Percentage of mineralization relative to control of cells after being treated with 25–200 ng/ml for 24 h. (D) Immunoblotting analysis of RUNX2 and osteocalcin, and (E) Quantitative expression levels of RUNX2 and osteocalcin after being treated with 25 and 50 ng/ml of CGRP peptide for 1 day. Data are expressed as mean ± SD(n = 5 for each group in triplicate); *p < 0.05 and **p < 0.01 compared with the control group, one-way ANOVA
Fig. 7
Fig. 7
CGRP peptides upregulates RAMP1 and activates p38. (A) Immunoblotting analysis of RAMP1 p38, and p-p38, and (B) Quantitative expression levels of RAMP1 p38, and p-p38, and (C) Expression ratio of p-p38/p38 after being treated with 25 and 50 ng/ml of CGRP peptide for 1 day. Data are expressed as mean ± SD(n = 5 for each group in triplicate); *p < 0.05 and **p < 0.01 compared with the control group, one-way ANOVA
Fig. 8
Fig. 8
RAMP1 possibly acts as an upstream regulator of p38 in response to osteoblast differentiation induced by compressive force. (A) Immunoblotting analysis of RAMP1, p38, p-p38, and RUNX2, (B) Quantitative expression levels of RAMP1, p38, p-p38, and RUNX2, and (C) Expression ratio of p-p38/p38 after being exposed to compressive forces (1–2 g/cm2) and compressive forces combined with 50 ng/ml CGRP. (D) Alizarin red staining of cells after being exposed to compressive forces (1–2 g/cm2) and compressive forces combined with 50 ng/ml CGRP. Data are expressed as mean ± SD(n = 5 for each group in triplicate); *p < 0.05 and **p < 0.01 compared with the control group, one-way ANOVA
Fig. 9
Fig. 9
Schematic diagram representing the mechanism underlying compressive force promoted osteoblast differentiation
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