Characterization of Thrombospondin Type 1 Repeat in Haliotis diversicolor and Its Possible Role in Osteoinduction.

Thrombospondin repeats (TSR) are important peptide domains present in the sequences of many extracellular and transmembrane proteins with which a variety of ligands interact. In this study, we characterized HdTSR domains in the ADAMTS3 protein of Thai abalone, Haliotis diversicolor, based on the transcriptomic analysis of its mantle tissues. PCR amplification and localization studies demonstrated the existence of HdTSR transcript and protein in H. diversicolor tissues, particularly in both the inner and outer mantle epithelial folds. We, therefore, generated a short recombinant protein, termed HdTSR1/2, based on the existence of the WxxWxxW or WxxxxW motif (which binds to TGF-ß, a known signaling in bone formation/repair) in HdTSR1 and HdTSR2 sequences and used it to test the osteoinduction function in the pre-osteoblastic cell line, MC3T3-E1. This recombinant protein demonstrated the ability to induce the differentiation of MC3T3-E1 cells by the concentration- and time-dependent upregulation of many known osteogenic markers, including RUNX2, COL1A1, OCN, and OPN. We also demonstrated the upregulation of the SMAD2 gene after cell treatment with HdTSR1/2 proteinindicating its possible interaction through TGF-ß, which thus activates its downstream signaling cascade and triggers the biomineralization process in the differentiated osteoblastic cells. Together, HdTSR domains existed in an extracellular ADAMTS3 protein in the mantle epithelium of H. diversicolor and played a role in osteoinduction as similar to the other nacreous proteins, opening up its possibility to be developed as an inducing agent of bone repair.

miR-130a and miR-27b Enhance Osteogenesis in Human Bone Marrow Mesenchymal Stem Cells via Specific Down-Regulation of Peroxisome Proliferator-Activated Receptor γ.

Mesenchymal stem cell (MSC) is a type of stem cell that is capable of differentiating into osteoblasts and adipocytes. The pathological perturbation of MSC fate determination is well demonstrated by the replacement of bone tissues with fat in those with osteoporosis and osteopenia. Cell fate determination can be regulated by epigenetic and post-transcriptional mechanisms. MicroRNAs (miRNAs) are small endogenous non-coding RNA molecules that mediates the post-transcriptional regulation of genes expression. We hypothesized that miRNA specified to PPARγ, a major transcription factor of adipogenesis, is responsible for the differentiation of MSCs into osteoblasts. Candidate miRNA that is responsible for target gene inhibition was identified from the miRNA database via bioinformatic analyses. In this study, miR-130a and miR-27b were selected for investigation on their role in specifically binding to peroxisome proliferator-activated receptor γ (PPARγ) via in vitro osteogenesis of human MSCs. During osteogenic differentiation of human MSCs, the expression level of miR-130a and miR-27b were found to be upregulated. In the meanwhile, adipogenic marker genes (PPARγ and C/EBPß) were found to decrease, which is in contrary to the increased expression of osteogenic marker genes (RUNX2 and Osterix). MSCs were transfected with mimics and inhibitors of miR-130a and miR-27b during in vitro osteogenesis followed by evaluation for the presence of osteogenic markers via quantitative gene expression, Western blot analysis and alkaline phosphatase activity assay. The overexpression of miR-130a and miR-27b is shown to enhance osteogenesis by increasing the gene expression of RUNX2 and Osterix, the protein expression of RUNX2, COL1A1, and Osterix as well as the alkaline phosphatase activity. Taken altogether, these results suggested that miR-130a and miR-27b could promote osteogenesis in human MSCs by targeting the PPARγ.

Exploring the epigenetic drug discovery landscape.

INTRODUCTION: Epigenetic modification has been implicated in a wide range of diseases and the ability to modulate such systems is a lucrative therapeutic strategy in drug discovery. Areas covered: This article focuses on the concepts and drug discovery aspects of epigenomics. This is achieved by providing a survey of the following concepts: (i) factors influencing epigenetics, (ii) diseases arising from epigenetics, (iii) epigenetic enzymes as druggable targets along with coverage of existing FDA-approved drugs and pharmacological agents, and (iv) drug repurposing/repositioning as a means for rapid discovery of pharmacological agents targeting epigenetics. Expert opinion: Despite significant interests in targeting epigenetic modifiers as a therapeutic route, certain classes of target proteins are heavily studied while some are less characterized. Thus, such orphan target proteins are not yet druggable with limited report of active modulators. Current research points towards a great future with novel drugs directed to the many complex multifactorial diseases of humans, which are still often poorly understood and difficult to treat.

Computational identification of miRNAs that modulate the differentiation of mesenchymal stem cells to osteoblasts.

MicroRNAs (miRNAs) are small endogenous noncoding RNAs that play an instrumental role in post-transcriptional modulation of gene expression. Genes related to osteogenesis (i.e., RUNX2, COL1A1 and OSX) is important in controlling the differentiation of mesenchymal stem cells (MSCs) to bone tissues. The regulated expression level of miRNAs is critically important for the differentiation of MSCs to preosteoblasts. The understanding of miRNA regulation in osteogenesis could be applied for future applications in bone defects. Therefore, this study aims to shed light on the mechanistic pathway underlying osteogenesis by predicting miRNAs that may modulate this pathway. This study investigates RUNX2, which is a major transcription factor for osteogenesis that drives MSCs into preosteoblasts. Three different prediction tools were employed for identifying miRNAs related to osteogenesis using the 3'UTR of RUNX2 as the target gene. Of the 1,023 miRNAs, 70 miRNAs were found by at least two of the tools. Candidate miRNAs were then selected based on their free energy values, followed by assessing the probability of target accessibility. The results showed that miRNAs 23b, 23a, 30b, 143, 203, 217, and 221 could regulate the RUNX2 gene during the differentiation of MSCs to preosteoblasts.

Osteoporosis: the current status of mesenchymal stem cell-based therapy.

Osteoporosis, or bone loss, is a progressive, systemic skeletal disease that affects millions of people worldwide. Osteoporosis is generally age related, and it is underdiagnosed because it remains asymptomatic for several years until the development of fractures that confine daily life activities, particularly in elderly people. Most patients with osteoporotic fractures become bedridden and are in a life-threatening state. The consequences of fracture can be devastating, leading to substantial morbidity and mortality of the patients. The normal physiologic process of bone remodeling involves a balance between bone resorption and bone formation during early adulthood. In osteoporosis, this process becomes imbalanced, resulting in gradual losses of bone mass and density due to enhanced bone resorption and/or inadequate bone formation. Several growth factors underlying age-related osteoporosis and their signaling pathways have been identified, such as osteoprotegerin (OPG)/receptor activator of nuclear factor B (RANK)/RANK ligand (RANKL), bone morphogenetic protein (BMP), wingless-type MMTV integration site family (Wnt) proteins and signaling through parathyroid hormone receptors. In addition, the pathogenesis of osteoporosis has been connected to genetics. The current treatment of osteoporosis predominantly consists of antiresorptive and anabolic agents; however, the serious adverse effects of using these drugs are of concern. Cell-based replacement therapy via the use of mesenchymal stem cells (MSCs) may become one of the strategies for osteoporosis treatment in the future.