Recent Progress in the Research on RNA-Binding Proteins in Bone Development and Diseases.

RNA-binding proteins (RBPs), which regulate gene expression through post-transcriptional modifications of RNAs, play a role in diverse biological processes that include bone cell development and bone tissue formation. RBP dysregulation may result in aberrant bone homeostasis and contribute to various bone diseases. The function of RBPs in bone physiology and pathophysiology and the underlying molecular mechanisms have been extensively studied in recent years. This article provides a review of such studies, highlighting the potential of RBPs as pivotal targets for therapeutic intervention.

Royal Jelly Enhances the Ability of Myoblast C2C12 Cells to Differentiate into Multilineage Cells.

Royal jelly (RJ) is recognized as beneficial to mammalian health. Multilineage differentiation potential is an important property of mesenchymal stem cells (MSCs). C2C12 cells have an innate ability to differentiate into myogenic cells. Like MSCs, C2C12 cells can also differentiate into osteoblast- and adipocyte-lineage cells. We recently reported that RJ enhances the myogenic differentiation of C2C12 cells. However, the effect of RJ on osteoblast or adipocyte differentiation is still unknown. Here in this study, we have examined the effect of RJ on the osteoblast and adipocyte differentiation of C2C12 cells. Protease-treated RJ was used to reduce the adverse effects caused by RJ supplementation. To induce osteoblast or adipocyte differentiation, cells were treated with bone morphogenetic proteins (BMP) or peroxisome proliferator-activated receptor γ (PPARγ) agonist, respectively. RNA-seq was used to analyze the effect of RJ on gene expression. We found that RJ stimulates osteoblast and adipocyte differentiation. RJ regulated 279 genes. RJ treatment upregulated glutathione-related genes. Glutathione, the most abundant antioxidative factor in cells, has been shown to promote osteoblast differentiation in MSC and MSC-like cells. Therefore, RJ may promote osteogenesis, at least in part, through the antioxidant effects of glutathione. RJ enhances the differentiation ability of C2C12 cells into multiple lineages, including myoblasts, osteoblasts, and adipocytes.

Osteocyte-mediated mechanical response controls osteoblast differentiation and function.

Low bone mass is a pervasive global health concern, with implications for osteoporosis, frailty, disability, and mortality. Lifestyle factors, including sedentary habits, metabolic dysfunction, and an aging population, contribute to the escalating prevalence of osteopenia and osteoporosis. The application of mechanical load to bone through physical activity and exercise prevents bone loss, while sufficient mechanical load stimulates new bone mass acquisition. Osteocytes, cells embedded within the bone, receive mechanical signals and translate these mechanical cues into biological signals, termed mechano-transduction. Mechano-transduction signals regulate other bone resident cells, such as osteoblasts and osteoclasts, to orchestrate changes in bone mass. This review explores the mechanisms through which osteocyte-mediated response to mechanical loading regulates osteoblast differentiation and bone formation. An overview of bone cell biology and the impact of mechanical load will be provided, with emphasis on the mechanical cues, mechano-transduction pathways, and factors that direct progenitor cells toward the osteoblast lineage. While there are a wide range of clinically available treatments for osteoporosis, the majority act through manipulation of the osteoclast and may have significant disadvantages. Despite the central role of osteoblasts to the deposition of new bone, few therapies directly target osteoblasts for the preservation of bone mass. Improved understanding of the mechanisms leading to osteoblastogenesis may reveal novel targets for translational investigation.

Galacto-oligosaccharide preconditioning improves metabolic activity and engraftment of Limosilactobacillus reuteri and stimulates osteoblastogenesis ex vivo.

A probiotic-related benefit for the host is inherently linked to metabolic activity and integration in the gut ecosystem. To facilitate these, probiotics are often combined with specific prebiotics in a synbiotic formulation. Here, we propose an approach for improving probiotic metabolic activity and engraftment. By cultivating the probiotic strain in the presence of a specific prebiotic (preconditioning), the bacterial enzymatic machinery is geared towards prebiotic consumption. Today, it is not known if preconditioning constitutes an advantage for the synbiotic concept. Therefore, we assessed the effects galacto-oligosaccharide (GOS) addition and preconditioning on GOS of Limosilactobacillus reuteri DSM 17938 on ex vivo colonic metabolic profiles, microbial community dynamics, and osteoblastogenesis. We show that adding GOS and preconditioning L. reuteri DSM 17938 act on different scales, yet both increase ex vivo short-chain fatty acid (SCFA) production and engraftment within the microbial community. Furthermore, preconditioned supernatants or SCFA cocktails mirroring these profiles decrease the migration speed of MC3T3-E1 osteoblasts, increase several osteogenic differentiation markers, and stimulate bone mineralization. Thus, our results demonstrate that preconditioning of L. reuteri with GOS may represent an incremental advantage for synbiotics by optimizing metabolite production, microbial engraftment, microbiome profile, and increased osteoblastogenesis.

Hsa-miR-15b-5p/miR-195-5p Controls Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells Through Regulating Indian Hedgehog Expression.

Osteoblastogenesis is regulated by several signaling pathways like hedgehog signaling. Of three types of mammalian Hedgehog genes, the Indian Hedgehog (Ihh) plays an important role in the formation of the skeleton. Mesenchymal stem cells (MSCs) isolated from adipose tissue have been considered a good source of osteoblast differentiation. Evidence also suggests that miRNAs play an important role in regulating key stages of osteoblast differentiation. In this study, two miRNAs targeting the Ihh were predicted by using bioinformatics analysis. ASCs were successfully derived, purified, and characterized from human adipose tissue. ASCs were chemically induced into osteoblast cells. Then, differentiation was confirmed by alkaline phosphatase (ALP) activity and Alizarin red staining. The relative expression of Ihh and related miRNAs was evaluated after 0, 7, 14, and 21 from the differentiation duration. The results of bioinformatics data showed that has-miR-195-5p and has-miR-15b-5p target the Ihh gene. The expression of Ihh significantly increased in a time-dependent manner in the differentiation process. In contrast, miR-195-5p and miR-15b-5p were significantly downregulated dependent on time duration (P < 0.01). Overall, the data indicate the antithetical regulation of Ihh versus has-miR-195-5p and has-miR-15b-5p during the differentiation process. These results support the hypothesis that these mi-RNAs could target the Ihh in the pathway of osteoblast differentiation derived from human ASCs.

Thyroid hormone receptor Thra and Thrb knockout differentially affects osteoblast biology and thyroid hormone responsiveness in vitro.

Thyroid hormones (TH) are important modulators of bone remodeling and thus, thyroid diseases, in particular hyperthyroidism, are able to compromise bone quality and fracture resistance. TH actions on bone are mediated by the thyroid hormone receptors (TR) TRα1 and TRß1, encoded by Thra and Thrb, respectively. Skeletal phenotypes of mice lacking Thra (Thra0/0 ) and Thrb (Thrb-/- ) are well-described and suggest that TRα1 is the predominant mediator of TH actions in bone. Considering that bone cells might be affected by systemic TH changes seen in these mutant mice, here we investigated the effects of TR knockout on osteoblasts exclusively at the cellular level. Primary osteoblasts obtained from Thra0/0 , Thrb-/- , and respective wildtype (WT) mice were analyzed regarding their differentiation potential, activity and TH responsiveness in vitro. Thra, but not Thrb knockout promoted differentiation and activity of early, mature and late osteoblasts as compared to respective WT cells. Interestingly, while mineralization capacity and expression of osteoblast marker genes and TH target gene Klf9 was increased by TH in WT and Thra-deficient osteoblasts, Thrb knockout mitigated the responsiveness of osteoblasts to short (48 h) and long term (10 d) TH treatment. Further, we found a low ratio of Rankl, a potent osteoclast stimulator, over osteoprotegerin, an osteoclast inhibitor, in Thrb-deficient osteoblasts and in line, supernatants obtained from Thrb-/- osteoblasts reduced numbers of primary osteoclasts in vitro. In accordance to the increased Rankl/Opg ratio in TH-treated WT osteoblasts only, supernatants from these cells, but not from TH-treated Thrb-/- osteoblasts increased the expression of Trap and Ctsk in osteoclasts, suggesting that osteoclasts are indirectly stimulated by TH via TRß1 in osteoblasts. In conclusion, our study shows that both Thra and Thrb differentially affect activity, differentiation and TH response of osteoblasts in vitro and emphasizes the importance of TRß1 to mediate TH actions in bone.

Effect of Ishophloroglucin A Isolated from Ishige okamurae on In Vitro Osteoclastogenesis and Osteoblastogenesis.

The balance between bone-resorbing osteoclasts and bone-forming osteoblasts is essential for the bone remodeling process. This study aimed to investigate the effect of Ishophloroglucin A (IPA) isolated from Ishige okamurae on the function of osteoclasts and osteoblasts in vitro. First, we demonstrated the effect of IPA on osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW 264.7 cells. IPA inhibited the tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation in RANKL-induced RAW 264.7 cells. Moreover, it inhibited the RANKL-induced osteoclast-related factors, such as TRAP, matrix metalloproteinase-9 (MMP-9), and calcitonin receptor (CTR), and transcription factors, such as nuclear factor of activated T cells 1 (NFATc1) and c-Fos. IPA significantly suppressed RANKL-activated extracellular signal-regulated kinase (ERK), and NF-κB in RAW 264.7 cells. Our data indicated that the ERK and NF-κB pathways were associated with the osteoclastogenesis inhibitory activity of IPA. Next, we demonstrated the effect of IPA on osteoblastogenesis in MG-63 cells. IPA significantly promoted alkaline phosphatase (ALP) activity in MG-63 cells, along with the osteoblast differentiation-related markers bone morphogenetic protein 2 (BMP2), type 1 collage (COL1), p-Smad1/5/8, and Runx2, by activating the MAPK signaling pathways. Taken together, the study indicated that IPA could be effective in treating bone diseases, such as osteoporosis.

Investigating the role of ASCC1 in the causation of bone fragility.

Bi-allelic variants in ASCC1 cause the ultrarare bone fragility disorder "spinal muscular atrophy with congenital bone fractures-2" (SMABF2). However, the mechanism by which ASCC1 dysfunction leads to this musculoskeletal condition and the nature of the associated bone defect are poorly understood. By exome sequencing, we identified a novel homozygous deletion in ASCC1 in a female infant. She was born with severe muscular hypotonia, inability to breathe and swallow, and virtual absence of spontaneous movements; showed progressive brain atrophy, gracile long bones, very slender ribs, and a femur fracture; and died from respiratory failure aged 3 months. A transiliac bone sample taken postmortem revealed a distinct microstructural bone phenotype with low trabecular bone volume, low bone remodeling, disordered collagen organization, and an abnormally high bone marrow adiposity. Proteomics, RNA sequencing, and qPCR in patient-derived skin fibroblasts confirmed that ASCC1 was hardly expressed on protein and RNA levels compared with healthy controls. Furthermore, we demonstrate that mutated ASCC1 is associated with a downregulation of RUNX2, the master regulator of osteoblastogenesis, and SERPINF1, which is involved in osteoblast and adipocyte differentiation. It also exerts an inhibitory effect on TGF-ß/SMAD signaling, which is important for bone development. Additionally, knockdown of ASCC1 in human mesenchymal stromal cells (hMSCs) suppressed their differentiation capacity into osteoblasts while increasing their differentiation into adipocytes. This resulted in reduced mineralization and elevated formation of lipid droplets. These findings shed light onto the pathophysiologic mechanisms underlying SMABF2 and assign a new biological role to ASCC1 acting as an important pro-osteoblastogenic and anti-adipogenic regulator.

Erythromycin Restores Osteoblast Differentiation and Osteogenesis Suppressed by Porphyromonas gingivalis Lipopolysaccharide.

The macrolide erythromycin (ERM) inhibits excessive neutrophil accumulation and bone resorption in inflammatory tissues. We previously reported that the expression of developmental endothelial locus-1 (DEL-1), an endogenous anti-inflammatory factor induced by ERM, is involved in ERM action. Furthermore, DEL-1 is involved in the induction of bone regeneration. Therefore, in this study, we investigated whether ERM exerts an osteoblastogenic effect by upregulating DEL-1 under inflammatory conditions. We performed in vitro cell-based mechanistic analyses and used a model of Porphyromonas gingivalis lipopolysaccharide (LPS)-induced periodontitis to evaluate how ERM restores osteoblast activity. In vitro, P. gingivalis LPS stimulation suppressed osteoblast differentiation and bone formation. However, ERM treatment combined with P. gingivalis LPS stimulation upregulated osteoblast differentiation-related factors and Del1, indicating that osteoblast differentiation was restored. Alveolar bone resorption and gene expression were evaluated in a periodontitis model, and the results confirmed that ERM treatment increased DEL-1 expression and suppressed bone loss by increasing the expression of osteoblast-associated factors. In conclusion, ERM restores bone metabolism homeostasis in inflammatory environments possibly via the induction of DEL-1.

Differential Expression of Non-Coding RNAs in Stem Cell Development and Therapeutics of Bone Disorders.

Stem cells' self-renewal and multi-lineage differentiation are regulated by a complex network consisting of signaling factors, chromatin regulators, transcription factors, and non-coding RNAs (ncRNAs). Diverse role of ncRNAs in stem cell development and maintenance of bone homeostasis have been discovered recently. The ncRNAs, such as long non-coding RNAs, micro RNAs, circular RNAs, small interfering RNA, Piwi-interacting RNAs, etc., are not translated into proteins but act as essential epigenetic regulators in stem cells' self-renewal and differentiation. Different signaling pathways are monitored efficiently by the differential expression of ncRNAs, which function as regulatory elements in determining the fate of stem cells. In addition, several species of ncRNAs could serve as potential molecular biomarkers in early diagnosis of bone diseases, including osteoporosis, osteoarthritis, and bone cancers, ultimately leading to the development of new therapeutic strategies. This review aims to explore the specific roles of ncRNAs and their effective molecular mechanisms in the growth and development of stem cells, and in the regulation of osteoblast and osteoclast activities. Furthermore, we focus on and explore the association of altered ncRNA expression with stem cells and bone turnover.

Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis.

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors. Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane-bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1-MMP are all expressed on the surface of RANKL-primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1-MMP. When TACE and MT1-MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti-TACE-mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC-sup) suppressed osteoblastogenesis from MC3T3-E1 cells, as measured by alkaline phosphatase activity, but OC-sup harvested from the osteoclast precursors treated with anti-TACE-mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti-TACE-mAb downregulated the generation of sSema4D in the mouse model of critical-sized bone defect, whereas local injection of recombinant sSema4D to anti-TACE-mAb-treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE-mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis.

Influence of Cholesterol on the Regulation of Osteoblast Function.

Bone is a dynamic tissue composed of cells, an extracellular matrix, and mineralized portion. Osteoblasts are responsible for proper bone formation and remodeling, and function. These processes are endergonic and require cellular energy in the form of adenosine triphosphate (ATP), which is derived from various sources such as glucose, fatty acids, and amino acids. However, other lipids such as cholesterol have also been found to play a critical role in bone homeostasis and can also contribute to the overall bioenergetic capacity of osteoblasts. In addition, several epidemiological studies have found a link between elevated cholesterol, cardiovascular disease, an enhanced risk of osteoporosis, and increased bone metastasis in cancer patients. This review focuses on how cholesterol, its derivatives, and cholesterol-lowering medications (statins) regulate osteoblast function and bone formation. It also highlights the molecular mechanisms underlying the cholesterol-osteoblast crosstalk.

Chinese Ecliptae herba (Eclipta prostrata (L.) L.) extract and its component wedelolactone enhances osteoblastogenesis of bone marrow mesenchymal stem cells via targeting METTL3-mediated m6A RNA methylation.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese Ecliptae herba (Eclipta prostrata (L.) L.) is an ethnomedicinal herb, which is used mainly to nourish kidney and thus strengthen bones according to traditional Chinese medicine theory. Pharmacological studies have supported the ethnomedicine use, showing that Ecliptae herba extract has an anti-osteoporotic effect in vivo and promoted osteoblast proliferation and activity in vitro. However, the molecular mechanism of Ecliptae herba on osteoblast differentiation from bone marrow mesenchymal stem cells (BMSC), the progenitors of osteoblasts, is still unclear. AIM OF THE STUDY: N6-methyladenosine (m6A) mRNA epigenetic modification may play a key role in promoting osteoblastic differentiation, and thus treating osteoporosis. This study sought to assess the mechanism through which Eclipate herba and its component wedelolactone influence m6A modification during the process of osteoblastogenesis from BMSC. MATERIAL AND METHODS: The alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were applied to determine osteoblastogenesis from BMSC. Western blot and quantitative real-time PCR were performed. RNA sequencing analysis was used to determine the characteristics of m6A methylation. Stable knocking down of METTL3 using lentiviral-based shRNA was performed. RESULTS: Upon 9 d treatment of BMSC with ethyl acetate extract of Ecliptae herba (MHL), ALP activity and ossification level increased in comparison with osteogenic medium (OS)-treated control. The expression of methyltransferase METTL3 and METTL14 was significantly increased, but WTAP expression had no change in response to MHL treatment. Knocking down of METTL3 resulted in a decrease in MHL-induced ALP activity, ossification level as well as mRNA expression of Osterix and Osteocalcin, two bone formation-related markers. The level of m6A increased when BMSC was treated with MHL for 9 d. RNA sequencing analysis indicated that MHL treatment altered mRNA m6A modification of genes associated with osteoblastogenesis. By kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, HIF-1α, PI3K/Akt, and Hippo signaling pathways were enriched and associated with m6A modification. The expression of m6A-modified genes including HIF-1α, VEGF-A, and RASSF1, was upregulated by MHL, but the upregulation was reversed after METTL3 knockdown. Additionally, the enhanced expression of METTL3 was also observed after treatment with wedelolactone, a component from MHL. CONCLUSIONS: These results suggested a previously uncharacterized mechanism of MHL and wedelolactone on osteoblastogenesis, by which METTL3-mediated m6A methylation is involved and thus contributes to the enhancement of osteoblastogenesis.

A novel implant surface modification mode of Fe3O4-containing TiO2 nanorods with sinusoidal electromagnetic field for osteoblastogenesis and angiogenesis.

Implants made of Ti and its alloys are widely utilized in orthopaedic surgeries. However, insufficient osseointegration of the implants often causes complications such as aseptic loosening. Our previous research discovered that disordered titanium dioxide nanorods (TNrs) had satisfactory antibacterial properties and biocompatibility, but TNrs harmed angiogenic differentiation, which might retarded the osseointegration process of the implants. Magnetic nanomaterials have a certain potential in promoting osseointegration, electromagnetic fields within a specific frequency and intensity range can facilitate angiogenic and osteogenic differentiation. Therefore, this study used Fe3O4 to endow magnetism to TNrs and explored the regulation effects of Ti, TNrs, and Fe3O4-TNrs under 1 â€‹mT 15 â€‹Hz sinusoidal electromagnetic field (SEMF) on osteoblastogenesis, osseointegration, angiogenesis, and its mechanism. We discovered that after the addition of SEMF treatment to VR-EPCs cultured on Fe3O4-TNrs, the calcineurin/NFAT signaling pathway was activated, which then reversed the inhibitory effect of Fe3O4-TNrs on angiogenesis. Besides, Fe3O4-TNrs with SEMF enhanced osteogenic differentiation and osseointegration. Therefore, the implant modification mode of Fe3O4-TNrs with the addition of SEMF could more comprehensively promote osseointegration and provided a new idea for the modification of implants.

DNA methylation analysis identifies key transcription factors involved in mesenchymal stem cell osteogenic differentiation.

BACKGROUND: Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. RESULTS: The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression. CONCLUSIONS: In this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.

Regulation of bone and fat balance by Fructus Ligustri Lucidi in ovariectomized mice.

CONTEXT: Fructus Ligustri Lucidi (FLL), a commonly used herb of traditional Chinese medicine (TCM), is the fruit of Ligustrum lucidum Ait. (Oleaceae). The ethanol extract of FLL is a potential candidate for preventing and treating postmenopausal osteoporosis (PMOP) by nourishing the liver and kidneys. OBJECTIVE: This study determines whether an ethanol extract of FLL has anti-osteoporotic effects in ovariectomized (OVX) mice and explores the underlying mechanism. MATERIALS AND METHODS: The OVX model of eight-week-old C57BL/6J female mice was taken, and ovariectomy was used as PMOP. Mice were divided into five groups: sham-operated group (n = 10), OVX group (n = 10), OVX + E2 group (n = 10; 0.039 mg/kg), OVX + FLL group (n = 10; 2 g/kg) and OVX + FLL group (n = 10; 4 g/kg). Mice were treated by gavage with FLL or CMCNa once daily for 8 weeks. We harvested uteri, femur, and tibias from mice; bone mineral density (BMD) and bone microstructure were obtained by X-ray absorptiometry and micro-CT. Furthermore, the effect of FLL on the balance of osteoblast and adipocyte differentiation was investigated using bone marrow mesenchymal stem cells (BMMSCs). RESULTS: The results indicated that FLL did not affect OVX-induced estradiol reduction. Compared with OVX mice, FLL significantly increased BMD (63.54 vs. 61.96), Conn. D (86.46 vs. 57.00), and left tibial strength (13.91 vs. 11.27), decreased Tb. Sp (0.38 vs. 0.44) and body fat content (4.19% vs. 11.24%). FLL decreased osteoclast activity and enhanced RUNX2 expression; inhibited perilipin peroxisome proliferator-activated receptor gamma (PPARγ) expression and adipocyte differentiation from BMMSCs. CONCLUSIONS: FLL prevented additional bone loss and improved bone microstructure in OVX mice by modulating bone and fat balance, suggesting that FLL might be a therapeutic agent for PMOP.

SPARC is a decoy counterpart for c­Fos and is associated with osteoblastic differentiation of bone marrow stromal cells by inhibiting adipogenesis.

Secreted protein acidic and rich in cysteine (SPARC), also called basement­membrane protein 40 or osteonectin, is a matricellular protein that is abundant not only in bone tissue as a non­collagenous protein but is also ubiquitously expressed in non­calcified tissue. SPARC is located intracellularly and disruption of the Sparc gene has been reported to reduce bone formation and increase fat tissue; however, the mechanism by which SPARC inhibits adipogenesis remains unclear. The present study evaluated the intracellular function of SPARC in adipogenesis using the bone marrow stromal cell line ST2. When ST2 cells with low SPARC production were cloned, intrinsic activator protein­1 (AP­1) activity was markedly higher, mineralized nodule formation was significantly lower and lipid accumulation was significantly increased compared with in the parental ST2 cells. Forced expression of secreted SPARC with the signal peptide­coding sequences of wild­type Sparc or preprotrypsin in SPARC­low ST2 cells significantly reduced AP­1 transcription activity; however, these reductions were not observed in the absence of signal peptide sequences. Recombinant SPARC, produced using Brevibacillus brevis, specifically bound to c­Fos but not c­Jun and inhibited the binding of c­Fos/c­Jun to a TPA­response element sequence. These data suggested that SPARC was incorporated into the cells from the extracellular spaces and serves an intracellular role as a decoy counterpart for c­Fos, as well as being associated with osteoblastogenesis through the inhibition of adipogenesis. These findings may provide new insights into regenerative medicine.

DNA methylation analysis identifies key transcription factors involved in mesenchymal stem cell osteogenic differentiation

BACKGROUND: Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. RESULTS: The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression. CONCLUSIONS: In this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.

Osteoblastogenesis of adipose-derived mesenchymal stem cells in chronic kidney disease patient with regular hemodialysis.

Background: Chronic kidney disease (CKD) is a health problem associated with high morbidity and mortality. Mineral and bone disorders are complications of CKD with a risk of fractures and cardiovascular disease. Mesenchymal stem cells can differentiate into osteoblasts and regulate their regulation by a network of cytokines and transcription factors. Objective: Analyzing differences in osteoblastogenesis of adipose mesenchymal stem cells in CKD patients and healthy people. Methods: The study sample was adipose mesenchymal stem cells from CKD patient undergoing hemodialysis and healthy people. Osteoblastogenesis was assessed by measuring the concentrations of transforming growth factor-ß1 (TGF-ß1), bone morphogenetic protein-2 (BMP-2), and (DKK-1) in culture media. The Elisa method measured the concentration of these parameters on days 4, 7, 14, and 21. Data were analyzed using an independent t-test and post hoc test with p-value <0.05. Result: There was a significant difference in CKD patients with increasing TGF-ß1 on day 4 (t = 2.821; 95% CI = 30,498-199,727; p = 0.010) and decreased on day 14. In the BMP-2 parameter, there was an increase on day 7 (t = 4.291; 95% CI = 0.289-0.831; p <0.001). Similar conditions were also found in the DKK-1 parameter, increasing on the 7th day, but there was no significant difference (p = 0.583). Conclusion: Osteoblastogenesis in adipose mesenchymal stem cells in CKD patients differs from that in healthy individuals. Osteoblasts fail in maturation and cause failure in matrix mineralization.

Kefir peptides ameliorate osteoporosis in AKR1A1 knockout mice with vitamin C deficiency by promoting osteoblastogenesis and inhibiting osteoclastogenesis.

The AKR1A1 protein is a member of the aldo-keto reductase superfamily that catalyzes the transformation of D-glucuronate to L-gulonate in the synthesis of L-ascorbic acid (vitamin C, Vit C). We previously demonstrated that AKR1A1 knockout mice (AKR1A1eGFP/eGFP) with Vit C deficiency exhibited aberrant bone formation and osteoporosis. In this study, we aimed to evaluate the osteoprotective effects of kefir peptides (KPs) in AKR1A1eGFP/eGFP mice and uncover the underlying mechanism of KPs in the modulation of bone remodeling. Six male CD-1 mice and 24 male AKR1A1eGFP/eGFP mice were used in this study, in which the AKR1A1eGFP/eGFP mice were randomly divided into four groups (n = 6). KPs treatment for 12 weeks exerted several effects in AKR1A1eGFP/eGFP mice including the reduction of serum proinflammatory cytokines (IL-1ß, IL-6, TNF-α), bone resorption markers (CTX-1, RANKL), and the increase of serum bone formation markers (P1NP, OPG, OC). µ-CT analysis indicated that KPs prevented the bone loss in the femurs of AKR1A1eGFP/eGFP mice by significantly increasing the trabecular parameters of bone mineral density, bone volume and bone number. Nanoindentation analysis demonstrated that KPs enhanced the elasticity and hardness of femoral cortical bones in AKR1A1eGFP/eGFP mice. KPs promoted bone marrow mesenchymal stem cells (BMMSCs)-derived osteoblast differentiation and mineralization by upregulating positive regulators of osteoblastogenesis (Runx2, ß-catenin, BMP-2, NFATc1). Conversely, KPs inhibited bone marrow macrophages (BMMs)-derived osteoclast differentiation and bone resorption, which was demonstrated by the facts that KPs suppressed RANKL-induced p38, NF-κB, Akt, PLCγ2 and CREB-1 phosphorylation, decreased the nuclear translocation of NFATc1 and c-Fos. Our findings demonstrate the efficacy of KPs in the prevention of osteoporosis in AKR1A1eGFP/eGFP mice and also unveil the dual effects of KPs in osteogenic promotion and osteoclastic inhibition. This study supports the use of KPs as nutritional supplements for the prevention of osteoporosis.