AIB1, a member of the steroid receptor coactivator (SRC) family that participates in gene transcriptional activation by nuclear receptors and other transcription factors, is required for animal growth and reproductive development and implicated in breast carcinogenesis. The mechanisms underlying the AIB1 pleiotropic functions are not fully understood and neither is the regulation of its activity. Here, we showed that AIB1 was a sumoylated protein and the sumoylation attenuated the transactivation activity of AIB1, which is in contrast to the sumoylation of its paralogs, GRIP1 and SRC-1. The transactivation activity of AIB1 is enhanced by its phosphorylation by several kinases, including mitogen-activated protein kinase. We demonstrated in this report that estrogen treatment led to an increased phosphorylation and decreased sumoylation of AIB1 and that the sumoylation coordinated with phosphorylation in regulating the transcriptional activity of AIB1, providing a mechanism for post-translational modifications in regulating the transcriptional output of AIB1.Nuclear receptors play critical roles in animal development and homeostasis through their bimodal function as repressors or activators of gene transcription (1). The repression and activation activity of nuclear receptors are determined by their association with corepressors and coactivators, respectively. Among the coactivators, the steroid receptor coactivator (SRC) 2 family is both necessary and sufficient for nuclear receptor-mediated gene activation as we previously demonstrated (2). The SRC family of coactivators includes three distinct but related p160 members: SRC-1, GRIP1 (or SRC-2), and AIB1 (also named RAC3, ACTR, SRC-3, or p/CIP in mice) (3-6). They share 40% overall sequence similarity, and they all contain, in the N terminus, a basic helix-loop-helix domain that is known for protein dimerization-DNA interaction as well as a Per-Arnt-Sim domain, which is known to be involved in protein-protein interaction. Three LXXLL motifs, also known as nuclear receptor boxes (for nuclear receptor binding), are centrally located in all three proteins (7-9). In the C terminus, the SRC proteins contain two activation domains, AD1 and AD2, which are associated with histone acetyltransferases (CREB-binding protein/p300 and pCAF) and coactivator-associated arginine methyltransferase 1, respectively, and function to modify the configuration of the chromatin structure (10, 11).Despite the structural similarities among the members of the SRC family of coactivators, evidence has been accumulated to suggest different physiological functions for SRC-1, GRIP1, and AIB1. Genetic studies with gene ablation showed that, although both male and female SRC1-null mice are viable and fertile, they exhibit partial resistance to several hormones, including estrogen, progestin, androgen, and thyroid hormones (12, 13). Elimination of GRIP1 impairs fertility in both male and female mice (14), and GRIP-null mice are protected against obesity and display enhanced adaptive thermogenesis, ...

Obesity is a risk factor for thoracic ossification of ligament flavum (TOLF) that is characterized by ectopic bone formation in the spinal ligaments. Hyperleptinemia is a common feature of obese people, and leptin, an adipocyte-derived cytokine with proliferative and osteogenic effects in several cell types, is believed to be an important factor in the pathogenesis of TOLF. However, how leptin might stimulate cell osteogenic differentiation in TOLF is not totally understood. We reported here that leptin-induced osteogenic effect in TOLF cells is associated with activation of signaling molecules STAT3, JNK, and ERK1/2 but not p38. Blocking STAT3 phosphorylation with a selective inhibitor, AG490, significantly abolished leptin-induced osteogenic differentiation of TOLF cells, whereas blocking ERK1/2 and JNK phosphorylation with their selective inhibitors PD98059 and SP600125, respectively, had only marginal effects. In addition, we showed that STAT3 interacted with Runt-related transcription factor 2 (Runx2) in the nucleus, and STAT3, Runx2, and steroid receptor coactivator steroid receptor coactivator-1 were components of the transcription complex recruited on Runx2 target gene promoters in response to leptin treatment. Our experiments identified STAT3, Runx2, and steroid receptor coactivator-1 as critical molecules in mediating leptin-stimulated cell osteogenesis in TOLF. Ossification of ligament flavum (OLF)3 of the spine is characterized by a heterotopic bone formation in the ligament flavum that is normally composed of fibrous tissues (1). Ossification could enlarge the spinal canal and compresses the spinal cord, resulting in serious neurological damages. Epidemiology has shown that high incidence rate of OLF occurs in thoracic spine (2). It has been documented that obesity represents a risk factor for thoracic ossification of ligament flavum (TOLF), particularly in Asian people (3). Indeed, hereditary obese rats, Zucker fatty (fa/fa) rats, are prone to OLF (4). A common feature of obese people is hyperleptinemia (5). Leptin, an adipocyte-derived cytokine, can stimulate the proliferation and osteogenic differentiation of various cell lines, such as the embryonic cell line C3H10T1/2, human NHOst cells, and human osteoblastic cells (6, 7). However, the molecular mechanism underlying the osteogenic effect of leptin in TOLF is not totally understood.Leptin exerts its biological activity through binding to its receptors, which belong to cytokine receptor superfamily. Different leptin receptor isoforms exist, including a long form (ObRb) and a short form (ObRa) (8). In vitro and in vivo studies have shown that leptin activates cytokine-like signal transduction via the long form receptor. Upon leptin stimulation, intracellular Janus tyrosine kinases are activated via transphosphorylation and phosphorylate tyrosine residues on the long form leptin receptor and on signal transducers and activators of transcription (STAT) proteins (9). Phosphorylated STAT proteins dimerize and translocate to the nucleus to activate ge...

The magnetic field has been proven to enhance bone tissue repair by affecting cell metabolic behavior. Magnetic nanoparticles are used as biomaterials due to their unique magnetic properties and good biocompatibility. Through endocytosis, entering the cell makes it easier to affect the physiological function of the cell. Once the magnetic particles are exposed to an external magnetic field, they will be rapidly magnetized. The magnetic particles and the magnetic field work together to enhance the effectiveness of their bone tissue repair treatment. This article reviews the common synthesis methods, the mechanism, and application of magnetic nanomaterials in the field of bone tissue repair.

Mechanical stress is considered to be an important factor in the progression of thoracic ossification of the ligament flavum (TOLF). To elucidate the mechanism underlying mechanical stress-induced TOLF, we investigated the effect of stretching on cultured flavum ligament cells derived from TOLF and non-TOLF patients. We found that the mRNA expression of alkaline phosphatase (ALP), osteocalcin, Runx2, and osterix, but not that of Dlx5 and Msx2, was significantly increased by stretching in TOLF cells. In addition, the effect seems to be finely tuned by stretching-triggered activation of distinct mitogen-activated protein kinase cascades. Specifically, a p38 specific inhibitor, SB203580, significantly inhibited stretching-induced osterix expression as well as ALP activity, whereas a specific inhibitor of ERK1/2, U0126, prevented stretching-induced Runx2 expression. We showed that overexpression of osterix resulted in a significant increase of ALP activity in TOLF cells, and osterix-specific RNAi completely abrogated the stretching-induced ALP activity, indicating that osterix plays a key role in stretching-stimulated osteogenic effect in TOLF cells. These results suggest that mechanical stress plays important roles in the progression of TOLF through induction of osteogenic differentiation of TOLF cells, and our findings support that osterix functions as a molecular link between mechanostressing and osteogenic differentiation.

Ossification of the ligamentum flavum (OLF) is a disorder of heterotopic ossification of spinal ligaments and is the main cause of thoracic spinal canal stenosis. Previous studies suggested that miR-132-3p negatively regulates osteoblast differentiation. However, whether miR-132-3p is involved in the process of OLF has not been investigated. In this study, we investigated the effect of miR-132-3p and its target genes forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6) on the osteogenic differentiation of ligamentum flavum (LF) cells. We demonstrated that miR-132-3p was down-regulated during the osteogenic differentiation of LF cells and negatively regulated the osteoblast differentiation. Further, miR-132-3p targeted FOXO1, GDF5 and SOX6 and down-regulated the protein expression of these genes. Meanwhile, FOXO1, GDF5 and SOX6 were up-regulated after osteogenic differentiation and the down-regulation of endogenous FOXO1, GDF5 or SOX6 suppressed the osteogenic differentiation of LF cells. In addition, we also found FOXO1, GDF5 and SOX6 expression in the ossification front of OLF samples. Overall, these results suggest that miR-132-3p inhibits the osteogenic differentiation of LF cells by targeting FOXO1, GDF5 and SOX6.

Mechanical stress and genetic factors play important roles in the occurrence of thoracic ossification of ligament flavum (TOLF), which can occur at one, two, or multiple levels of the spine. It is unclear whether single- and multiple-level TOLF differ in terms of osteogenic differentiation potency and osteogenesis-related gene expression under cyclic mechanical stress. This was addressed in the present study using patients with non-TOLF and single- and multiple-level TOLF (n=8 per group). Primary ligament cells were cultured and osteogenesis was induced by application of cyclic mechanical stress. Osteogenic differentiation was assessed by evaluating alkaline phosphatase (ALP) activity and the mRNA and protein expression of osteogenesis-related genes, including ALP, bone morphogenetic protein 2 (BMP2), Runt-related transcription factor-2 (Runx-2), osterix, osteopontin (OPN) and osteocalcin. The application of cyclic mechanical stress resulted in higher ALP activity in the multiple-level than in the single-level TOLF group, whereas no changes were observed in the non-TOLF group. The ALP, BMP2, OPN and osterix mRNA levels were higher in the multiple-level as compared to the single-level TOLF group, and the levels of all osteogenesis-related genes, apart from Runx2, were higher in the multiple-level as compared to the non-TOLF group. The osterix and ALP protein levels were higher in the multiple-level TOLF group than in the other 2 groups, and were increased with the longer duration of stress. These results highlight the differences in osteogenic differentiation potency between single- and multiple-level TOLF that may be related to the different pathogenesis and genetic background.

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This study investigated the pathological process of Notch signaling in the osteogenesis of ligamentum flavum tissues and cells, and the associated regulatory mechanisms. Notch receptors, ligands, and target genes were identified by quantitative polymerase chain reaction (qPCR) in ligamentum flavum cells and immunohistochemistry in ligamentum flavum sections from ossification of the ligamentum flavum (OLF) patients and controls. The temporospatial expression patterns of JAG1/Notch2/HES1 in human ligamentum flavum cells during osteogenic differentiation were determined by qPCR. Lentiviral vectors for Notch2 overexpression and knockdown were constructed and transfected into ligamentum flavum cells before osteogenic differentiation to examine the function of Notch signaling pathways in the osteogenic differentiation of ligamentum flavum cells. Alkaline phosphatase, Runx2, Osterix, osteocalcin, and osteopontin mRNA levels, alkaline phosphatase activity, and Alizarin Red staining were used as indicators of osteogenic differentiation. JAG1/Notch2/ HES1 mRNA levels were up-regulated in ligamentum flavum cells from OLF patients, which increased during osteogenic differentiation. Immunohistochemical analysis suggested positive Notch2 expression at the ossification front. Down-regulation of Notch2 expression decelerated osteogenic differentiation of ligamentum flavum cells, and Notch2 overexpression promoted osteogenic differentiation of ligamentum flavum cells. Expression of Runx2 and Osterix increased in a manner similar to that of Notch2 during osteogenic differentiation of ligamentum flavum cells, and Notch2 knockdown and overexpression influenced their expression levels. Notch signaling plays an important role in OLF, and Notch may affect the osteogenic differentiation of ligamentum flavum cells via interactions with Runx2 and Osterix.ß