IADR Abstract Archives
RAMP1 Regulates Osteogenic Differentiation of BMSCs by Yap1
Objectives: Recently, it has been suggested that receptor activity-modifying protein-1 (RAMP1) might be a critical regulator of calcitonin gene-related peptide-alpha (αCGRP) mediated effects during bone wound healing. However, the roles and mechanisms of RAMP1 on osteogenesis remain unclear. This study aims to provide the reader with a novel insight of RAMP1 and explore the effects of Yap1, an effector of Hippo pathway, with this process.
Methods: BMSCs were collected from femoral and tibial bone marrow of male mice. After that, we first applied RAMP1 overexpression lentiviral vector system and stably transfected it into BMSCs. Western blot was employed to confirm the expression of RAMP1. CCK8 assay was monitored to examine the effects of lentiviral vectors on cell proliferation. To further uncover the effects of RAMP1 on osteogenic differentiation, verteporfin was used to block the downstream Yap1 signaling. Alkaline phosphatase (ALP) activity assessment and mineralization staining were performed. In addition, the expression of osteoblastic marker genes was evaluated at mRNA levels.
Results: BMSCs transfected with RAMP1 overexpression lentiviral vector confirmed strongest RAMP1 protein expression than the controls. CCK8 assay further showed that lentiviral vectors had negligible adverse effects on cell proliferation. According to real-time PCR results, expression levels of ALP, COL-1, OCN were upregulated in RAMP1 overexpression group (p<0.05). Moreover, RAMP1-transfected BMSCs exhibited increased ALP activity and more mineralized nodules. Notably, verteporfin could impair RAMP1 induced osteogenesis.
Conclusions: Our findings demonstrate that a recombinant lentiviral RAMP1 expression vector is a promising strategy to explore the effects and molecular mechanisms of RAMP1. RAMP1 overexpression could promote osteogenic differentiation of BMSCs and Yap1 is involved in this process. RAMP1 might be a potential therapeutic target in the repair of alveolar bone fracture healing or insufficient bone mass.
Methods: BMSCs were collected from femoral and tibial bone marrow of male mice. After that, we first applied RAMP1 overexpression lentiviral vector system and stably transfected it into BMSCs. Western blot was employed to confirm the expression of RAMP1. CCK8 assay was monitored to examine the effects of lentiviral vectors on cell proliferation. To further uncover the effects of RAMP1 on osteogenic differentiation, verteporfin was used to block the downstream Yap1 signaling. Alkaline phosphatase (ALP) activity assessment and mineralization staining were performed. In addition, the expression of osteoblastic marker genes was evaluated at mRNA levels.
Results: BMSCs transfected with RAMP1 overexpression lentiviral vector confirmed strongest RAMP1 protein expression than the controls. CCK8 assay further showed that lentiviral vectors had negligible adverse effects on cell proliferation. According to real-time PCR results, expression levels of ALP, COL-1, OCN were upregulated in RAMP1 overexpression group (p<0.05). Moreover, RAMP1-transfected BMSCs exhibited increased ALP activity and more mineralized nodules. Notably, verteporfin could impair RAMP1 induced osteogenesis.
Conclusions: Our findings demonstrate that a recombinant lentiviral RAMP1 expression vector is a promising strategy to explore the effects and molecular mechanisms of RAMP1. RAMP1 overexpression could promote osteogenic differentiation of BMSCs and Yap1 is involved in this process. RAMP1 might be a potential therapeutic target in the repair of alveolar bone fracture healing or insufficient bone mass.