Detecting the Mechanism behind the Transition from Fixed Two-Dimensional Patterned Sika Deer (Cervus nippon) Dermal Papilla Cells to Three-Dimensional Pattern.

The hair follicle dermal papilla is critical for hair generation and de novo regeneration. When cultured in vitro, dermal papilla cells from different species demonstrate two distinguishable growth patterns under the conventional culture condition: a self-aggregative three dimensional spheroidal (3D) cell pattern and a two dimensional (2D) monolayer cell pattern, correlating with different hair inducing properties. Whether the loss of self-aggregative behavior relates to species-specific differences or the improper culture condition remains unclear. Can the fixed 2D patterned dermal papilla cells recover the self-aggregative behavior and 3D pattern also remains undetected. Here, we successfully constructed the two growth patterns using sika deer (Cervus nippon) dermal papilla cells and proved it was the culture condition that determined the dermal papilla growth pattern. The two growth patterns could transit mutually as the culture condition was exchanged. The fixed 2D patterned sika deer dermal papilla cells could recover the self-aggregative behavior and transit back to 3D pattern, accompanied by the restoration of hair inducing capability when the culture condition was changed. In addition, the global gene expressions during the transition from 2D pattern to 3D pattern were compared to detect the potential regulating genes and pathways involved in the recovery of 3D pattern and hair inducing capability.

Dynamic consolidated bioprocessing for innovative lab-scale production of bacterial alkaline phosphatase from Bacillus paralicheniformis strain APSO.

To meet the present and forecasted market demand, bacterial alkaline phosphatase (ALP) production must be increased through innovative and efficient production strategies. Using sugarcane molasses and biogenic apatite as low-cost and easily available raw materials, this work demonstrates the scalability of ALP production from a newfound Bacillus paralicheniformis strain APSO isolated from a black liquor sample. Mathematical experimental designs including sequential Plackett-Burman followed by rotatable central composite designs were employed to select and optimize the concentrations of the statistically significant media components, which were determined to be molasses, (NH4)2NO3, and KCl. Batch cultivation in a 7-L stirred-tank bioreactor under uncontrolled pH conditions using the optimized medium resulted in a significant increase in both the volumetric and specific productivities of ALP; the alkaline phosphatase throughput 6650.9 U L-1, and µ = 0.0943 h-1; respectively, were obtained after 8 h that, ameliorated more than 20.96, 70.12 and 94 folds compared to basal media, PBD, and RCCD; respectively. However, neither the increased cell growth nor enhanced productivity of ALP was present under the pH-controlled batch cultivation. Overall, this work presents novel strategies for the statistical optimization and scaling up of bacterial ALP production using biogenic apatite.

Phosphocreatine Promotes Osteoblastic Activities in H2O2-Induced MC3T3-E1 Cells by Regulating SIRT1/FOXO1/PGC-1α Signaling Pathway.

BACKGROUND: Osteoporosis, characterized by bone loss, usually occurs with the increased bone resorption and decreased bone formation. H2O2-induced MC3T3-E1 cells are commonly used for the study of osteoblastic activities, which play a crucial role in bone formation. OBJECTIVE: This study aimed to investigate the effects of Phosphocreatine (PCr) on the osteoblastic activities in H2O2-induced MC3T3-E1 cells and elaborate on the possible molecular mechanism. METHODS: The Osteoprotegerin (OPG)/Receptor Activator of NF-κB Ligand (RANKL) ratio and osteogenic markers were detected to investigate the effects of PCr on osteoblastic activities, and the osteoblastic apoptosis was detected using Hochest staining. Moreover, oxidative stress, Adenosine Triphosphate (ATP) generation and the expression of Sirtuin 1 (SIRT1), Forkhead Box O 1 (FOXO1) and Peroxisome Proliferator-Activated Receptor Γ Coactivator-1α (PGC-1α) were also examined to uncover the possible molecular mechanism in H2O2-induced MC3T3-E1 cells. RESULT: The results showed that PCr promoted the osteoblastic differentiation by increasing the expression levels of osteogenic markers of Alkaline Phosphatase (ALP) and Runt-related transcription factor 2 (Runx2), as well as increased the OPG/RANKL ratio and suppressed the osteoblastic apoptosis in H2O2-induced MC3T3-E1 cells. Moreover, treatment with PCr suppressed reactive oxygen species (ROS) over-generation and promoted the ATP production as well as increased the PGC-1α, FOXO1 and SIRT1 protein expression levels in H2O2-induced MC3T3-E1 cells. CONCLUSION: PCr treatment could promote osteoblastic activities via suppressing oxidative stress and increasing the ATP generation in H2O2-induced MC3T3-E1 cells. In addition, the positive effects of PCr on osteoblasts might be regulated by SIRT1/FOXO1/ PGC-1α signaling pathway.

High Erythropoiesis Resistance Index Is a Significant Predictor of Cardiovascular and All-Cause Mortality in Chinese Maintenance Hemodialysis Patients.

BACKGROUND: Renal anemia is a common complication of hemodialysis patients. Erythropoietin (EPO) hyporesponsiveness has been recognized as an important factor to poor efficacy of recombinant human erythropoietin in the treatment of renal anemia. More importantly, increased erythropoiesis resistance index (ERI) may be associated with inflammation and increased mortality. OBJECTIVE: The objective of this research was to investigate correlated factors of EPO responsiveness and to clarify the relationships between EPO hyporesponsiveness and cardiovascular mortality and all-cause mortality among maintenance hemodialysis patients. METHODS: This prospective cohort study enrolled 276 maintenance hemodialysis patients for a 55-month follow-up to investigate the factors related to ERI and its relationship to all-cause mortality and cardiovascular mortality. RESULTS: ERI was positively correlated with predialysis serum high-sensitivity C-reactive protein (r = 0.234, p < 0.001), alkaline phosphatase (r = 0.134, p = 0.028), and ferritin (r = 0.155, p = 0.010) and negatively correlated with albumin (r = -0.206, p < 0.001) and creatinine (r = -0.232, p < 0.001). As multiple linear regression showed, predialysis serum albumin, high-sensitivity C-reactive protein, ferritin, and creatinine were independent correlated factors of ERI (p < 0.05). Kaplan-Meier curves showed that the cumulative incidences of both cardiovascular mortality and all-cause mortality were significantly higher in patients with ERI > 11.04 IU/kg/w/g/dL (both p < 0.01). The high ERI group was significantly associated with higher risk for all-cause mortality (OR 1.781, 95% CI 1.091 to 2.910, p = 0.021) and cardiovascular mortality (OR 1.972, 95% CI 1.139 to 3.417, p = 0.015) after adjusting for confounders. CONCLUSIONS: Predialysis serum albumin, high-sensitivity C-reactive protein, ferritin, and creatinine were independent correlated factors of EPO responsiveness among maintenance hemodialysis patients. Patients with higher ERI values had a higher all-cause mortality rate and cardiovascular mortality rate.

Astrocytes in the Ventrolateral Preoptic Area Promote Sleep.

Although ventrolateral preoptic (VLPO) nucleus is regarded as a center for sleep promotion, the exact mechanisms underlying the sleep regulation are unknown. Here, we used optogenetic tools to identify the key roles of VLPO astrocytes in sleep promotion. Optogenetic stimulation of VLPO astrocytes increased sleep duration in the active phase in naturally sleep-waking adult male rats (n = 6); it also increased the extracellular ATP concentration (n = 3) and c-Fos expression (n = 3-4) in neurons within the VLPO. In vivo microdialysis analyses revealed an increase in the activity of VLPO astrocytes and ATP levels during sleep states (n = 4). Moreover, metabolic inhibition of VLPO astrocytes reduced ATP levels (n = 4) and diminished sleep duration (n = 4). We further show that tissue-nonspecific alkaline phosphatase (TNAP), an ATP-degrading enzyme, plays a key role in mediating the somnogenic effects of ATP released from astrocytes (n = 5). An appropriate sample size for all experiments was based on statistical power calculations. Our results, taken together, indicate that astrocyte-derived ATP may be hydrolyzed into adenosine by TNAP, which may in turn act on VLPO neurons to promote sleep.SIGNIFICANCE STATEMENT Glia have recently been at the forefront of neuroscience research. Emerging evidence illustrates that astrocytes, the most abundant glial cell type, are the functional determinants for fates of neurons and other glial cells in the central nervous system. In this study, we newly identified the pivotal role of hypothalamic ventrolateral preoptic (VLPO) astrocytes in the sleep regulation, and provide novel insights into the mechanisms underlying the astrocyte-mediated sleep regulation.

A simvastatin-releasing scaffold with periodontal ligament stem cell sheets for periodontal regeneration.

Simvastatin (SIM) has been documented to induce the osteogenic differentiation of periodontal ligament stem cells (PDLSCs). To establish an efficient release system for periodontal regeneration, a polycaprolactone (PCL) membrane scaffold containing SIM was electrospun and evaluated. The obtained PCL-SIM membrane scaffold showed sustained release up to 28 days, without deleterious effect on proliferation of PDLSCs on the scaffolds. PDLSCs were seeded onto scaffolds and their osteogenic differentiation was evaluated. After 21 days, expressions of collagen type I, alkaline phosphatase and bone sialoprotein genes were significantly upregulated and mineralized matrix formation was increased on the PCL-SIM scaffolds compared with the PCL scaffolds. In a heterotopic periodontal regeneration model, a cell sheet-scaffold construct was assembled by placement of multilayers of PDLSC sheets on PCL or PCL-SIM scaffolds, and these were then placed between dentin and ceramic bovine bone for subcutaneous implantation in athymic mice. After 8 weeks, the PCL-SIM membrane showed formation of significantly more ectopic cementum-like mineral on the dentin surface. These findings demonstrated that the PCL-SIM membrane scaffold promotes cementum-like tissue formation by sustained drug release, suggesting the feasibility of its therapeutic use with PDLSC sheets to improve periodontal regeneration.

Investigation of alpl expression and Tnap-activity in zebrafish implies conserved functions during skeletal and neuronal development.

Hypophosphatasia (HPP) is a rare genetic disease with diverse symptoms and a heterogeneous severity of onset with underlying mutations in the ALPL gene encoding the ectoenzyme Tissue-nonspecific alkaline phosphatase (TNAP). Considering the establishment of zebrafish (Danio rerio) as a new model organism for HPP, the aim of the study was the spatial and temporal analysis of alpl expression in embryos and adult brains. Additionally, we determined functional consequences of Tnap inhibition on neural and skeletal development in zebrafish. We show that expression of alpl is present during embryonic stages and in adult neuronal tissues. Analyses of enzyme function reveal zones of pronounced Tnap-activity within the telencephalon and the mesencephalon. Treatment of zebrafish embryos with chemical Tnap inhibitors followed by axonal and cartilage/mineralized tissue staining imply functional consequences of Tnap deficiency on neuronal and skeletal development. Based on the results from neuronal and skeletal tissue analyses, which demonstrate an evolutionary conserved role of this enzyme, we consider zebrafish as a promising species for modeling HPP in order to discover new potential therapy strategies in the long-term.

Boosting toxic protein biosynthesis: transient in vivo inactivation of engineered bacterial alkaline phosphatase.

BACKGROUND: The biotechnology production of enzymes is often troubled by the toxicity of the recombinant products of cloned and expressed genes, which interferes with the recombinant hosts' metabolism. Various approaches have been taken to overcome these limitations, exemplified by tight control of recombinant genes or secretion of recombinant proteins. An industrial approach to protein production demands maximum possible yields of biosynthesized proteins, balanced with the recombinant host's viability. Bacterial alkaline phosphatase (BAP) from Escherichia coli (E. coli) is a key enzyme used in protein/antibody detection and molecular cloning. As it removes terminal phosphate from DNA, RNA and deoxyribonucleoside triphosphates, it is used to lower self-ligated vectors' background. The precursor enzyme contains a signal peptide at the N-terminus and is secreted to the E. coli periplasm. Then, the leader is clipped off and dimers are formed upon oxidation. RESULTS: We present a novel approach to phoA gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme. The recombinant bap gene was modified by replacing a secretion leader coding section with a N-terminal His6-tag, cloned and expressed in E. coli in a PBAD promoter expression vector. The gene expression was robust, resulting in accumulation of His6-BAP in the cytoplasm, exceeding 50% of total cellular proteins. The His6-BAP protein was harmless to the cells, as its natural toxicity was inhibited by the reducing environment within the E. coli cytoplasm, preventing formation of the active enzyme. A simple protocol based on precipitation and immobilized metal affinity chromatography (IMAC) purification yielded homogeneous protein, which was reactivated by dialysis into a redox buffer containing reduced and oxidized sulfhydryl group compounds, as well as the protein structure stabilizing cofactors Zn2+, Mg2+ and phosphate. The reconstituted His6-BAP exhibited high activity and was used to develop an efficient protocol for all types of DNA termini, including problematic ones (blunt, 3'-protruding). CONCLUSIONS: The developed method appears well suited for the industrial production of ultrapure BAP. Further, the method of transient inactivation of secreted toxic enzymes by conducting their biosynthesis in an inactive state in the cytoplasm, followed by in vitro reactivation, can be generally applied to other problematic proteins.

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli.

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.

Enzymatic characterization and regulation of gene expression of PhoK alkaline phosphatase in Sphingobium sp. strain TCM1.

Sphingobium sp. strain TCM1 can significantly degrade chlorinated organophosphorus flame retardants, such as tris(2-chloroethyl) phosphate. The PhoK of strain TCM1 (Sb-PhoK) is the main alkaline phosphatase (APase) that catalyzes the last step in the degradation pathway. Here, we purified and characterized Sb-PhoK produced in E. coli, and analyzed the regulation of Sb-phoK gene expression in strain TCM1. The recombinant Sb-PhoK was produced in the mature form, lacking a putative signal peptide, and formed a homodimer. Purified Sb-PhoK exhibited 384 U/mg of specific activity at 37 °C. The optimum temperature was 50 °C, and Sb-PhoK was completely inactivated when incubated at 60 °C for 10 min. The optimum pH was 10, with stability observed at pH 6.0-10.5. Sb-PhoK was suggested to contain two Ca2+ and one Zn2+ per subunit, but excess addition of Zn2+ into the reaction mixture markedly inhibited the enzyme activity. Sb-PhoK showed phosphatase activity against various phosphorylated compounds, except for bis(p-nitrophenyl) phosphate, indicating that it is a phosphomonoesterase with broad substrate specificity. The Km and kcat for p-nitrophenyl phosphate were 2.31 mM and 1270 s-1, respectively, under optimal conditions. The enzyme was strongly inhibited by vanadate, dithiothreitol, and SDS, but was highly resistant to urea and Triton X-100. Sb-phoK gene expression was regulated by the inorganic phosphate concentration in culture medium, and was induced at a low inorganic phosphate concentration. The deletion of Sb-phoB gene resulted in no induction of Sb-phoK gene even at a low inorganic phosphate concentration, confirming that Sb-PhoK is a member of Pho regulon.

Expression, Folding, and Activation of Halophilic Alkaline Phosphatase in Non-Halophilic Brevibacillus choshinensis.

Halophilic enzymes contain a large number of acidic amino acids and marginal large hydrophobic amino acids, which make them highly soluble even under strongly hydrophobic conditions. This characteristic of halophilic enzymes provides potential for their industrial application. However, halophilic enzymes easily degrade when used for industrial applications compared with enzymes from other extremophiles because of their instability in low-salt environments. We aimed to clarify the stabilization mechanism of halophilic enzymes. We previously attempted to express halophilic alkaline phosphatase from Halomonas (HaALP) in non-halophilic E. coli. However, the expressed HaALP showed little activity. Therefore, we overexpressed HaALP in Gram-positive non-halophilic Brevibacillus choshinensis in this study, which was successfully expressed and purified in its active form. HaALP was denatured in 6 M urea, refolded using various salts and the non-ionic osmolyte trimethylamine N-oxide (TMAO), and assessed by native polyacrylamide gel electrophoresis. HaALP refolded in 3 M NaCl or 3 M TMAO containing Na+ ions. Hydrophobic interactions due to a high salt concentration or TMAO enhanced the formation of the folding intermediate (the monomer precursor), and only Na+ ions activated the dimer form. This insight into the stabilization mechanism of HaALP may lead to the development of industrial applications of halophilic enzymes under low-salt conditions.

Characterization of five newly derived canine osteosarcoma cell lines.

BACKGROUND: Canine and human osteosarcomas (OS) are notably similar and have a high rate of metastasis. There is a poor understanding of the tumor development process, predisposing causes, and varying levels of aggression among different cell lines. By characterizing newly developed canine osteosarcoma cell lines, treatments for people and pets can be developed. Of the seven subtypes of OS, three are represented in this group: osteoblastic (the most common), fibroblastic, and giant cell variant. To our knowledge, there are no other giant cell variant canine OS cell lines in the published literature and only one canine fibroblastic osteosarcoma cell line. Understanding the differences between the histologic subtypes in dogs will help to guide comparative research. RESULTS: Alkaline phosphatase expression was ubiquitous in all cell lines tested and invasiveness was variable between the cell lines tested. Invasiveness and oxidative damage were not correlated with in vivo growth rates, where TOT grew the fastest and had the higher percentage of mice with metastatic lesions. TOL was determined to be the most chemo-resistant during cisplatin chemotherapy while TOM was the most chemo-sensitive. CONCLUSIONS: Further comparisons and studies using these cell lines may identify a variety of characteristics valuable for understanding the disease process and developing treatments for osteosarcoma in both species. Some of this data was presented as a poster by KMF at the August 5th, 2017 National Veterinary Scholars Program in Bethesda, MA. Characterization of 5 newly generated canine osteosarcoma cell lines. Kelli Franks, Tasha Miller, Heather Wilson-Robles.

Effect of Fluoride Doping in Laponite Nanoplatelets on Osteogenic Differentiation of Human Dental Follicle Stem Cells (hDFSCs).

Bioactive nanosilicates are emerging prominent next generation biomaterials due to their intrinsic functional properties such as advanced biochemical and biophysical cues. Recent studies show interesting dose-dependent effect of fluoride ions on the stem cells. Despite of interesting properties of fluoride ions as well as nanosilicate, there is no reported literature on the effect of fluoride-doped nanosilicates on stem cells. We have systematically evaluated the interaction of fluoride nanosilicate platelets (NS + F) with human dental follicle stem cells (hDFSCs) to probe the cytotoxicity, cellular transport (internalization) and osteogenic differentiation capabilities in comparison with already reported nanosilicate platelets without fluoride (NS - F). To understand the osteoinductive and osteoconductive properties of the nanosilicate system, nanosilicate treated hDFSCs are cultured in three different medium namely normal growth medium, osteoconductive medium, and osteoinductive medium up to 21 d. NS + F treated stem cells show higher ALP activity, osteopontin levels and significant alizarin red staining compared to NS - F treated cells. This study highlights that the particles having fluoride additives (NS + F) aid in enhancing the osteogenic differentiation capabilities of hDFSCs thus potential nanobiomaterial for periodontal bone tissue regeneration.

Considerations for high-yield, high-throughput cell enrichment: fluorescence versus magnetic sorting.

Efficient sorting methods are required for the isolation of cellular subpopulations in basic science and translational applications. Despite this, throughputs, yields, viabilities, and processing times of common sorting methods like fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) are underreported. In the current study, we set out to quantify the ability of these sorting methods to separate defined mixtures of alkaline phosphatase liver/bone/kidney (ALPL)-expressing and non-expressing cell types. Results showed that initial MACS runs performed using manufacturer's recommended antibody and microbead concentrations produced inaccurate ALPL+ vs. ALPL- cell splits compared to FACS when ALPL+ cells were present in larger proportions (>~25%). Accuracy at all proportions could be achieved by using substantially higher concentrations of labeling reagents. Importantly, MACS sorts resulted in only 7-9% cell loss compared to ~70% cell loss for FACS. Additionally, MACS processing was 4-6 times faster than FACS for single, low proportion samples but took similar time for single, high-proportion samples. When processing multiple samples, MACS was always faster overall due to its ability to run samples in parallel. Average cell viability for all groups remained high (>83%), regardless of sorting method. Despite requiring substantial optimization, the ability of MACS to isolate increased cell numbers in less time than FACS may prove valuable in both basic science and translational, cell-based applications.

Whole synthetic pathway engineering of recombinant protein production.

The output from protein biomanufacturing systems is a function of total host cell biomass synthetic capacity and recombinant protein production per unit cell biomass. In this study, we describe how these two properties can be simultaneously optimized via design of a product-specific combination of synthetic DNA parts to maximize flux through the protein synthetic pathway and the use of a host cell chassis with an increased capability to synthesize both cell and product biomass. Using secreted alkaline phosphatase (SEAP) production in Chinese hamster ovary cells as our example, we demonstrate how an optimal composition of input components can be assembled from a minimal toolbox containing rationally designed promoters, untranslated regions, signal peptides, product coding sequences, cell chassis, and genetic effectors. Product titer was increased 10-fold, compared with a standard reference system by (a) identifying genetic components that acted in concert to maximize the rates of SEAP transcription, translation, and translocation, (b) selection of a cell chassis with increased biomass synthetic capacity, and (c) engineering the host cell factory's capacity for protein folding and secretion. This whole synthetic pathway engineering process to design optimal expression cassette-chassis combinations should be applicable to diverse recombinant protein and host cell-type contexts.

Estrogen-mediated gut microbiome alterations influence sexual dimorphism in metabolic syndrome in mice.

BACKGROUND: Understanding the mechanism of the sexual dimorphism in susceptibility to obesity and metabolic syndrome (MS) is important for the development of effective interventions for MS. RESULTS: Here we show that gut microbiome mediates the preventive effect of estrogen (17ß-estradiol) on metabolic endotoxemia (ME) and low-grade chronic inflammation (LGCI), the underlying causes of MS and chronic diseases. The characteristic profiles of gut microbiome observed in female and 17ß-estradiol-treated male and ovariectomized mice, such as decreased Proteobacteria and lipopolysaccharide biosynthesis, were associated with a lower susceptibility to ME, LGCI, and MS in these animals. Interestingly, fecal microbiota-transplant from male mice transferred the MS phenotype to female mice, while antibiotic treatment eliminated the sexual dimorphism in MS, suggesting a causative role of the gut microbiome in this condition. Moreover, estrogenic compounds such as isoflavones exerted microbiome-modulating effects similar to those of 17ß-estradiol and reversed symptoms of MS in the male mice. Finally, both expression and activity of intestinal alkaline phosphatase (IAP), a gut microbiota-modifying non-classical anti-microbial peptide, were upregulated by 17ß-estradiol and isoflavones, whereas inhibition of IAP induced ME and LGCI in female mice, indicating a critical role of IAP in mediating the effects of estrogen on these parameters. CONCLUSIONS: In summary, we have identified a previously uncharacterized microbiome-based mechanism that sheds light upon sexual dimorphism in the incidence of MS and that suggests novel therapeutic targets and strategies for the management of obesity and MS in males and postmenopausal women.

Effects of silicate-based composite material on the proliferation and mineralization behaviors of human dental pulp cells: An in vitro assessment.

The objective of this study was to investigate the effects of a silicate-based composite material on proliferation and mineralization of human dental pulp cells (hDPCs), which was compared with those of calcium hydroxide (Ca(OH)2, CH) and tricalcium silicate (Ca3SiO5, C3S). HDPCs were cultured with CH, C3S and tricalcium silicate/dicalcium silicate (Ca3SiO5/Ca2SiO4, C3S/C2S) composites extract. The CCK-8 assay showed that the composite material stimulated the proliferation of hDPCs. The odontogenic marker genes and DSPP protein expression were more significantly up-regulated by the C3S/C2S composite material compared with pure CH and C3S. HDPCs cultured with composite material extract exert stronger ALP activity and alizarin red S staining. C3S/C2S composite material was advantageous over pure C3S by showing enhanced ability to stimulate the proliferation and odontogenic differentiation of hDPCs, suggesting that the C3S/C2S composite materials possess desirable biocompatibility and bioactivity, and might be a new type of pulp-capping agent and dentin alternative materials.

Dishevelled­2 modulates osteogenic differentiation of human synovial fibroblasts in osteoarthritis.

Dishevelled (Dvl)­2 represents one of the cytoplasmic proteins, which serves as a pivotal hub in signaling intermediates through a number of different signaling pathways associated with the Wnt family. The aim of the present study was to investigate the roles and mechanisms of Dvl­2 on synovial fibroblasts (SFBs) in osteoarthritis (OA). A Cell Counting kit­8 (CCK­8) assay was used to determine cell viability. An alkaline phosphatase (ALP) test kit was used to measure the activity of ALP. Western blot and reverse transcription­quantitative polymerase chain reaction analysis were used to evaluate the protein and mRNA expression, respectively. The results suggest that depletion of Dvl­2 significantly decreased the expression of osteoprotegerin (OPG) and ALP (P<0.05) and significantly increased the expression of receptor activator of nuclear factor­κB ligand (RANKL), ALP, osteonectin (ON), osteocalcin (OCN) and osterix (P<0.05). In addition, the depletion of Dvl­2 also significantly inhibited the expression of runt­related transcription factor 2 (Runx­2) and ß­catenin in SFBs (P<0.05). The effect of Dvl­2 over­expression was opposite to the effect of Dvl­2 silencing. The inactivation of Wnt3a reversed the effect of Dvl­2 silencing. In conclusion, the results indicate that Dvl­2 regulated osteogenic differentiation of SFBs in OA.

lncRNA HOTAIR Inhibits Mineralization in Osteoblastic Osteosarcoma Cells by Epigenetically Repressing ALPL.

HOTAIR is a lncRNA that plays critical role in gene regulation and chromatin dynamics through epigenetic mechanisms. In this work we studied the physiological role of HOTAIR during the process of mineralization using osteoblastic osteosarcoma cells focusing in ALPL (Tissue Non-Specific Alkaline Phosphatase), a pivotal gene that controls bone formation. HOTAIR knockdown resulted in upregulation of ALPL, increase of alkaline phosphatase (ALP) activity, and enhanced mineralization in osteoblastic SaOS-2 cells cultured in mineralizing medium. Luciferase assays using reporter vectors containing ALPL promoter showed that HOTAIR repression increases ALPL promoter activity. Furthermore, HOTAIR knockdown increased histone H3K4 methylation levels at ALPL promoter region, suggesting that ALPL repression by HOTAIR is regulated by epigenetic mechanisms. This work supports that physiological bone formation is epigenetically regulated by a lncRNA.

Regulation of Vascular Calcification by Growth Hormone-Releasing Hormone and Its Agonists.

RATIONALE: Vascular calcification (VC) is a marker of the severity of atherosclerotic disease. Hormones play important roles in regulating calcification; estrogen and parathyroid hormones exert opposing effects, the former alleviating VC and the latter exacerbating it. To date no treatment strategies have been developed to regulate clinical VC. OBJECTIVE: The objective of this study was to investigate the effect of growth hormone-releasing hormone (GHRH) and its agonist (GHRH-A) on the blocking of VC in a mouse model. METHODS AND RESULTS: Young adult osteoprotegerin-deficient mice were given daily subcutaneous injections of GHRH-A (MR409) for 4 weeks. Significant reductions in calcification of the aortas of MR409-treated mice were paralleled by markedly lower alkaline phosphatase activity and a dramatic reduction in the expression of transcription factors, including the osteogenic marker gene Runx2 and its downstream factors, osteonectin and osteocalcin. The mechanism of action of GHRH-A was dissected in smooth muscle cells isolated from human and mouse aortas. Calcification of smooth muscle cells induced by osteogenic medium was inhibited in the presence of GHRH or MR409, as evidenced by reduced alkaline phosphatase activity and Runx2 expression. Inhibition of calcification by MR409 was partially reversed by MIA602, a GHRH antagonist, or a GHRH receptor-selective small interfering RNA. Treatment with MR409 induced elevated cytosolic cAMP and its target, protein kinase A which in turn blocked nicotinamide adenine dinucleotide phosphate oxidase activity and reduced production of reactive oxygen species, thus blocking the phosphorylation of nuclear factor κB (p65), a key intermediate in the ligand of receptor activator for nuclear factor-κ B-Runx2/alkaline phosphatase osteogenesis program. A protein kinase A-selective small interfering RNA or the chemical inhibitor H89 abolished these beneficial effects of MR409. CONCLUSIONS: GHRH-A controls osteogenesis in smooth muscle cells by targeting cross talk between protein kinase A and nuclear factor κB (p65) and through the suppression of reactive oxygen species production that induces the Runx2 gene and alkaline phosphatase. Inflammation-mediated osteogenesis is thereby blocked. GHRH-A may represent a new pharmacological strategy to regulate VC.