Pin1-mediated Modification Prolongs the Nuclear Retention of β-Catenin in Wnt3a-induced Osteoblast Differentiation

The canonical Wnt signaling pathway, in which β-catenin nuclear localization is a crucial step, plays an important role in osteoblast differentiation. Pin1, a prolyl isomerase, is also known as a key enzyme in osteogenesis. However, the role of Pin1 in canonical Wnt signal-induced osteoblast differentiation is poorly understood. We found that Pin1 deficiency caused osteopenia and reduction of β-catenin in bone lining cells. Similarly, Pin1 knockdown or treatment with Pin1 inhibitors strongly decreased the nuclear β-catenin level, TOP flash activity, and expression of bone marker genes induced by canonical Wnt activation and vice versa in Pin1 overexpression. Pin1 interacts directly with and isomerizes β-catenin in the nucleus. The isomerized β-catenin could not bind to nuclear adenomatous polyposis coli, which drives β-catenin out of the nucleus for proteasomal degradation, which consequently increases the retention of β-catenin in the nucleus and might explain the decrease of β-catenin ubiquitination. These results indicate that Pin1 could be a critical target to modulate β-catenin-mediated osteogenesis.     

Age-related changes in bone formation,osteoblastic cell proliferation,and differentiation during postnatal osteogenesis in human calvaria

We have determined the age-related changes in the growth characteristics and expression of the osteoblast phenotype in human calvaria osteoblastic cells in relation with histologic indices of bone formation during postnatal calvaria osteogenesis. Histomorphometric analysis of normal calvaria samples obtained from 36 children, aged 3 to 18 months, showed an age-related decrease in the extent of bone surface covered with osteoblasts and newly synthesized collagen, demonstrating a progressive decline in bone formation during postnatal calvaria osteogenesis. Immunohistochemical analysis showed expression of type I collagen, bone sialoprotein, and osteonectin in the matrix and osteoblasts, with no apparent age-related change during postnatal calvaria osteogenesis. Cells isolated from human calvaria displayed characteristics of the osteoblast phenotype including alkaline phosphatase (ALP) activity, osteocalcin (OC) production, expression of bone matrix proteins, and responsiveness to calciotropic hormones. The growth of human calvaria osteoblastic cells was high at 3 months of age and decreased with age, as assessed by (³H)-thymidine incorporation into DNA. Thus, the age-related decrease in bone formation is associated with a decline in osteoblastic cell proliferation during human calvaria osteogenesis. In contrast, ALP activity and OC production increased with age in basal conditions and in response to 1,25(OH)₂, vitamin D₃, suggesting a reciprocal relationship between cell growth and expression of phenotypic markers during human postnatal osteogenesis. Finally, we found that human calvaria osteoblastic cells isolated from young individuals with high bone formation activity in vivo and high growth potential in vitro had the ability to form calcified nodular bone-like structures in vitro in the presence of ascorbic acid and β-glycerophosphate, providing a new model to study human osteogenesis in vitro. J. Cell. Biochem. 64:128–139. © 1997 Wiley-Liss, Inc.     

HuR-positive stress granules: Potential targets for age-related osteoporosis

Aging impairs osteoblast function and bone turnover, resulting in age-related bone degeneration. Stress granules (SGs) are membrane-less organelles that assemble in response to stress via the recruitment of RNA-binding proteins (RBPs), and have emerged as a novel mechanism in age-related diseases. Here, we identified HuR as a bone-related RBP that aggregated into SGs and facilitated osteogenesis during aging. HuR-positive SG formation increased during osteoblast differentiation, and HuR overexpression mitigated the reduction in SG formation observed in senescent osteoblasts. Moreover, HuR positively regulated the mRNA stability and expression of its target β-catenin by binding and recruiting β-catenin into SGs. As a potential therapeutic target, HuR activator apigenin (API) enhanced its expression and thus aided osteoblasts differentiation. API treatment increased HuR nuclear export, enhanced the recruitment of β-catenin into HuR-positive SGs, facilitated β-catenin nuclear translocation, and contributed osteogenesis. Our findings highlight the roles of HuR and its SGs in promoting osteogenesis during skeletal aging and lay the groundwork for novel therapeutic strategies against age-related skeletal disorders.     

Collagen type XV and the ‘osteogenic status’

We have previously demonstrated that collagen type XV (ColXV) is a novel bone extracellular matrix (ECM) protein. It is well known that the complex mixture of multiple components present in ECM can help both to maintain stemness or to promote differentiation of stromal cells following change in qualitative characteristics or concentrations. We investigated the possible correlation between ColXV expression and mineral matrix deposition by human mesenchymal stromal cells (hMSCs) with different osteogenic potential and by osteoblasts (hOBs) that are able to grow in culture medium with or without calcium. Analysing the osteogenic process, we have shown that ColXV basal levels are lower in cells less prone to osteo‐induction such as hMSCs from Wharton Jelly (hWJMSCs), compared to hMSCs that are prone to osteo‐induction such as those from the bone marrow (hBMMSCs). In the group of samples identified as ‘mineralized MSCs’, during successful osteogenic induction, ColXV protein continued to be detected at substantial levels until early stage of differentiation, but it significantly decreased and then disappeared at the end of culture when the matrix formed was completely calcified. The possibility to grow hOBs in culture medium without calcium corroborated the results obtained with hMSCs demonstrating that calcium deposits organized in a calcified matrix, and not calcium ‘per se’, negatively affected ColXV expression. As a whole, our data suggest that ColXV may participate in ECM organization in the early‐phases of the osteogenic process and that this is a prerequisite to promote the subsequent deposition of mineral matrix.     

Single molecule effects of osteogenesis imperfecta mutations in tropocollagen protein domains

Osteogenesis imperfecta (OI) is a genetic disease characterized by fragile bones, skeletal deformities and, in severe cases, prenatal death that affects more than 1 in 10,000 individuals. Here we show by full atomistic simulation in explicit solvent that OI mutations have a significant influence on the mechanical properties of single tropocollagen molecules, and that the severity of different forms of OI is directly correlated with the reduction of the mechanical stiffness of individual tropocollagen molecules. The reduction of molecular stiffness provides insight into the molecular‐scale mechanisms of the disease. The analysis of the molecular mechanisms reveals that physical parameters of side‐chain volume and hydropathy index of the mutated residue control the loss of mechanical stiffness of individual tropocollagen molecules. We propose a model that enables us to predict the loss of stiffness based on these physical characteristics of mutations. This finding provides an atomistic‐level mechanistic understanding of the role of OI mutations in defining the properties of the basic protein constituents, which could eventually lead to new strategies for diagnosis and treatment the disease. The focus on material properties and their role in genetic diseases is an important, yet so far only little explored, aspect in studying the mechanisms that lead to pathological conditions. The consideration of how material properties change in diseases could lead to a new paradigm that may expand beyond the focus on biochemical readings alone and include a characterization of material properties in diagnosis and treatment, an effort referred to as materiomics.     

The NH2-terminal and COOH-terminal fragments of dentin matrix protein 1 (DMP1) localize differently in the compartments of dentin and growth plate of bone

Multiple studies have shown that dentin matrix protein 1 (DMP1) is essential for bone and dentin mineralization. After post-translational proteolytic cleavage, DMP1 exists within the extracellular matrix of bone and dentin as an NH2-terminal fragment, a COOH-terminal fragment, and the proteoglycan form of the NH2-terminal fragment (DMP1-PG). To begin to assess the biological function of each fragment, we evaluated the distribution of both fragments in the rat tooth and bone using antibodies specific to the NH2-terminal and COOH-terminal regions of DMP1 and confocal microscopy. In rat first molar organs, the NH2-terminal fragment localized to predentin, whereas the COOH-terminal fragment was mainly restricted to mineralized dentin. In the growth plate of bone, the NH2-terminal fragment appeared in the proliferation and hypertrophic zones, whereas the COOH-terminal fragment occupied the ossification zone. Forster resonance energy transfer analysis showed colocalization of both fragments of DMP1 in odontoblasts and predentin, as well as hypertrophic chondrocytes within the growth plates of bone. The biochemical analysis of bovine teeth showed that predentin is rich in DMP1-PG, whereas mineralized dentin primarily contains the COOH-terminal fragment. We conclude that the differential patterns of expression of NH2-terminal and COOH-terminal fragments of DMP1 reflect their potentially distinct roles in the biomineralization of dentin and bone matrices.     

Twist2的生物学功能及其分子机制

碱性螺旋-环-螺旋(Basic helix-loop-helix protein,bHLH)家族成员Twist2对间质细胞系的发生和发育起转录调节作用,经直接或间接机制发挥分子开关功能,从而激活或抑制靶基因。Twist2能直接结合DNA上 E-box保守序列,招募共激活物或抑制剂;能与E蛋白调节因子发生蛋白-蛋白相互作用,干扰激活或抑制功能。Twist2无义突变导致Setleis综合征。对Twist2的早期研究多集中在骨骼发育,随后在多种肿瘤中发现其有表达差异,研究表明Twist2在肿瘤的上皮-间质转化(Epithelial-mesenchymal transition,EMT)中发挥着重要作用。Twist2参与了多条通路的调控,其调控作用的发挥受到时空表达、磷酸化、二聚化和细胞定位的调节,在机体的正常发育、体内平衡和疾病发生机制中研究Twist2的作用显得尤为重要。文章对Twist2在成骨分化、肿瘤形成和EMT中的作用及其分子机制进行综述,以便帮助了解Twist2的生物学功能,为进一步在疾病的诊断、发展、以及治疗等方面的转化应用研究提供依据。