Jeeranan Manokawinchoke1, Prasit Pavasant2, Thanaphum Osathanon3. 1. Mineralized Tissue Research Unit and Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: aorjm711@gmail.com. 2. Mineralized Tissue Research Unit and Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: prasit215@gmail.com. 3. Mineralized Tissue Research Unit and Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand; Craniofacial Genetics and Stem Cell Research Group, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: thanaphum.o@chula.ac.th.
Abstract
OBJECTIVE: Different mechanical stimuli regulate behaviors of various cell types, including osteoblasts, osteocytes, and periodontal ligament fibroblasts. Notch signaling participates in the mechanical stress-regulated cell responses. The present study investigated the regulation of Notch target gene and sclerostin (Sost) expression in murine pre-osteoblast cell line (MC3T3-E1) under intermittent compressive stress. METHODS: MC3T3-E1 were subjected to the intermittent compressive force under the computerized controlled machine. In some experiments, cells were pretreated with chemical inhibitors for Notch and transforming growth factor (TGF)-β signaling prior to mechanical stimuli. To evaluate role of Notch signaling in MC3T3-E1 cells under unloaded condition, cells were seeded on indirect immobilized Notch ligand (Jagged1). Gene expression was determined using real-time quantitative polymerase chain reaction. RESULTS: The intermittent compressive stress significantly upregulated Notch target gene expression (Hes Family BHLH transcription factor 1; Hes1 and Hairy/enhancer-of-split related with YRPW motif protein1; Hey1). The intermittent stress-induced Hes1 and Hey1 mRNA expression could be inhibited by a γ-secretase inhibitor (DAPT) or a TGF-β superfamily type I activing receptor-like kinase receptors inhibitor (SB431542). The results imply that intermittent compressive stress regulates Notch signaling via TGF-β pathway. Further, the intermittent compressive stress reduced Sost mRNA expression and this phenomenon could be rescued by a DAPT pretreatment, implying the involvement of Notch signaling. However, activation of Notch signaling under the unloaded condition resulted in the increase of Sost expression and the reduction of osteogenic marker genes. CONCLUSIONS: These results imply the involvement of Notch signaling in the homeostasis maintaining of osteogenic cells under mechanical stress stimuli.
OBJECTIVE: Different mechanical stimuli regulate behaviors of various cell types, including osteoblasts, osteocytes, and periodontal ligament fibroblasts. Notch signaling participates in the mechanical stress-regulated cell responses. The present study investigated the regulation of Notch target gene and sclerostin (Sost) expression in murine pre-osteoblast cell line (MC3T3-E1) under intermittent compressive stress. METHODS: MC3T3-E1 were subjected to the intermittent compressive force under the computerized controlled machine. In some experiments, cells were pretreated with chemical inhibitors for Notch and transforming growth factor (TGF)-β signaling prior to mechanical stimuli. To evaluate role of Notch signaling in MC3T3-E1 cells under unloaded condition, cells were seeded on indirect immobilized Notch ligand (Jagged1). Gene expression was determined using real-time quantitative polymerase chain reaction. RESULTS: The intermittent compressive stress significantly upregulated Notch target gene expression (Hes Family BHLH transcription factor 1; Hes1 and Hairy/enhancer-of-split related with YRPW motif protein1; Hey1). The intermittent stress-induced Hes1 and Hey1 mRNA expression could be inhibited by a γ-secretase inhibitor (DAPT) or a TGF-β superfamily type I activing receptor-like kinase receptors inhibitor (SB431542). The results imply that intermittent compressive stress regulates Notch signaling via TGF-β pathway. Further, the intermittent compressive stress reduced Sost mRNA expression and this phenomenon could be rescued by a DAPT pretreatment, implying the involvement of Notch signaling. However, activation of Notch signaling under the unloaded condition resulted in the increase of Sost expression and the reduction of osteogenic marker genes. CONCLUSIONS: These results imply the involvement of Notch signaling in the homeostasis maintaining of osteogenic cells under mechanical stress stimuli.