Collagen-derived dipeptide prolyl-hydroxyproline promotes osteogenic differentiation through Foxg1

Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. We previously reported that Pro-Hyp promotes the differentiation of osteoblasts by increasing Runx2, osterix and Col1α1 mRNA expression levels. Here, to elucidate the mechanism of Pro-Hyp promotion of osteoblast differentiation, we focus on the involvement of Foxo1 in osteoblast differentiation via Runx2 regulation and the role of Foxg1 in Foxo1 regulation. The addition of Pro-Hyp had no effect on MC3T3-E1 cell proliferation in Foxo1- or Foxg1-knockdown cells. In Foxo1-knockdown cells, the addition of Pro-Hyp increased ALP activity, but in Foxg1-knockdown cells, it had no effect on ALP activity. An enhancing effect of Pro-Hyp on the Runx2 and osterix expression levels was observed in Foxo1-knockdown cells. However, no enhancing effect of Pro-Hyp on osteoblastic gene expression was observed when Foxg1 was knocked down. These results demonstrate that Pro-Hyp promotes osteoblastic MC3T3-E1 cell differentiation and upregulation of osteogenic genes via Foxg1 expression.     

ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice

Osteogenic differentiation refers to the process of bone formation and remodeling, which is controlled by complex molecular mechanisms. Activin A receptor type I (ACVR1) is reported to be associated with osteogenic differentiation. However, the underlying molecular mechanism remains elusive. Therefore, this study evaluates the function of ACVR1 in osteogenic differentiation through the Wnt signaling pathway. The expression of osteocalcin (Oc) and osterix together with osteogenic differentiation and mineralization was examined in ACVR1-knockout (KO) mouse. Furthermore, the Wnt signaling pathway was inhibited in bone marrow stromal cells (BMSCs) of mice to explore the role of the Wnt signaling pathway in osteogenic differentiation by means of alkaline phosphatase (ALP) activity detection and evaluation of mineralized nodules and calcium content. Subsequently, the effect of ACVR1 on the Wnt signaling pathway was assessed by determining the expression of ACVR1, β-catenin, glycogen synthase kinase 3 β (GSK3β), dickkopf-related protein 1 (DKK1), and frizzled class receptor 1 (FZD1). Both their effects on osteogenic differentiation were further evaluated by determination of Oc, osterix, and Runx2 expression. AVCR1 KO mice exhibited increased Oc and osterix expression and promoted bone resorption and formation. ACVR1-knockout was observed to activate the Wnt signaling pathway with an increase of β-catenin and reductions in GSK3β, DKK1, and FZD1. With the inhibited Wnt signaling pathway expression of Oc, osterix, and Runx2 was decreased, and ALP activity, mineralized nodule, and calcium content in cellular matrix were decreased as well, indicating that inactivation of the Wnt signaling pathway reduced the differentiation of BMSCs into osteoclasts. These findings indicate that ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice.     

LGR5 regulates osteogenic differentiation of human thoracic ligamentum flavum cells by Wnt signalling pathway

Thoracic ossification of the ligamentum flavum (TOLF) is ectopic ossification of the spinal ligaments. Histologically, the development of TOLF can be described as the process of endochondral ossification. However, the underlying aetiology has not been completely clarified. In this investigation, the gene expression profile associated with leucine‐rich repeat‐containing G‐protein‐coupled receptors (LGR) and Wnt signalling pathway in the thoracic ligamentum flavum cells (TLFCs) of different ossification stages was analysed via RNA sequencing. We further confirmed the significant differences in the related gene expression profile by Gene Ontology (GO) enrichment analysis. LGR5 was first identified in primary human TLFCs during osteogenic differentiation. To evaluate the effect of LGR5 on osteogenic differentiation, LGR5 has been knocked down and overexpressed in human TLFCs. We observed that the knockdown of LGR5 inhibited the activity of Wnt signalling and attenuated the potential osteogenic differentiation of TLFCs, while overexpression of LGR5 activated the Wnt signalling pathway and increased osteogenic differentiation. Our results provide important evidence for the potent positive mediatory effects of LGR5 on osteogenesis by enhancing the Wnt signalling pathway in TOLF.     

CTHRC1 promotes osteogenic differentiation of periodontal ligament stem cells by regulating TAZ

Collagen triple helix repeat containing 1 (CTHRC1) is associated with bone metabolism. Alveolar bone has an ability to rapidly remodel itself to adapt its biomechanical environment and function. However, whether CTHRC1 is expressed in alveolar bone tissue and the role of CTHRC1 in alveolar bone remodeling remain unclear. We used orthodontic tooth movement (OTM) rat model to study the effects of CHTRC1 in alveolar bone remodeling in vivo. We found that CTHRC1 was expressed in normal physiological condition of osteocytes, bone matrix, and periodontal ligament cells in rat. During the OTM, the expression of CTHRC1, Runx2 and TAZ were increased. We further studied the effects of CTHRC1 on osteogenic differentiation of human periodontal ligament stem cells in vitro. CTHRC1 can positively regulate the expression of TAZ and osteogenic differentiation markers like Col1, ALP, Runx2 and OCN. Overexpression of CHTRC1 increased osteogenic differentiation of PDLSCs, which could be abolished by TAZ siRNA. Our results suggest that CTHRC1 plays an important role in alveolar bone remodeling and osteogenic differentiation of PDLSCs.     

Vitamin C induces periodontal ligament progenitor cell differentiation via activation of ERK pathway mediated by PELP1

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our fi nding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.     

Heparin inhibits BMP-2 osteogenic bioactivity by binding to both BMP-2 and BMP receptor

Heparin demonstrates several kinds of biological activities by binding to various extracellular molecules and plays pivotal roles in bone metabolism. However, the role of heparin in the biological activity of bone morphogenetic protein (BMP) remains unclear. In the present study, we examined whether heparin has the effects on osteoblast differentiation induced by BMP-2 in vitro and also elucidated the precise mechanism by which heparin regulates bone metabolism induced by this molecule. Our results showed that heparin inhibited alkaline phosphatase (ALP) activity and mineralization in osteoblastic cells cultured with BMP-2. Heparin was found to suppress the mRNA expressions of osterix, Runx2, ALP and osteocalcin, as well as phosphorylation of Smad1/5/8 and p38 MAPK. Further, heparin bound to both BMP-2 and BMP receptor (BMPR). These results suggest that heparin suppresses BMP-2-BMPR binding, and inhibits BMP-2 osteogenic activity in vitro.