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Sample records for connexin43 potentiates osteoblast

  1. Proliferation, differentiation and apoptosis in connexin43-null osteoblasts

    NASA Technical Reports Server (NTRS)

    Furlan, F.; Lecanda, F.; Screen, J.; Civitelli, R.

    2001-01-01

    Osteoblasts are highly coupled by gap junctions formed primarily by connexin43 (Cx43). We have shown that interference with Cx43 expression or function disrupts transcriptional regulation of osteoblast genes, and that deletion of Cx43 in the mouse causes skeletal malformations, delayed mineralization, and osteoblast dysfunction. Here, we studied the mechanisms by which genetic deficiency of Cx43 alters osteoblast development. While cell proliferation rates were similar in osteoblastic cells derived from calvaria of Cx43-null and wild type mice, camptothecin-induced apoptosis was 3-fold higher in mutant compared to wild type osteoblasts. When grown in mineralizing medium, Cx43-null cells were able to produce mineralized matrix but it took one week longer to reach the same mineralization levels as in normal cells. Likewise, expression of alkaline phosphatase activity per cell--a marker of osteoblast differentiation--was maximal only 2 weeks later in Cx43-null relative to wild-type cells. These observations suggest that Cx43 is important for a normal and timely development of the osteoblastic phenotype. Delayed differentiation and increase programmed cell death may explain the skeletal phenotype of Cx43-null mice.

  2. Connexin43 and Pannexin1 Channels in Osteoblasts: Who is the “hemichannel”?

    PubMed Central

    Thi, Mia M.; Islam, Shalena; Suadicani, Sylvia O.; Spray, David C.

    2012-01-01

    Osteoblasts sense and respond to mechanical stimuli in a process involving influx and release of large ions and signaling molecules. Unapposed gap junction hemichannels formed of connexin43 (Cx43) have been proposed as a major route for such exchange, and in particular for release of ATP and prostaglandin E2 (PGE2) in osteocytes. However, we have found that Cx43-null osteoblasts have unaltered mechanically-induced PGE2 release and ATP-induced YoPro dye uptake. In contrast, PGE2 release in response to fluid shear stress is abolished in P2X7 receptor (P2X7R) null osteoblasts and ATP-induced dye uptake is attenuated following treatment of wildtype cells with a P2X7R or Pannexin1 (Panx1) channel blocker. These data indicate that Panx1 channels, in concert with P2X7R, likely form a molecular complex that performs the hemichannel function in osteoblast mechanosignaling. PMID:22797941

  3. Low peak bone mass and attenuated anabolic response to parathyroid hormone in mice with an osteoblast-specific deletion of connexin43.

    PubMed

    Chung, Dong Jin; Castro, Charlles H M; Watkins, Marcus; Stains, Joseph P; Chung, Min Young; Szejnfeld, Vera Lucia; Willecke, Klaus; Theis, Martin; Civitelli, Roberto

    2006-10-15

    Connexin43 (Cx43) is involved in bone development, but its role in adult bone homeostasis remains unknown. To overcome the postnatal lethality of Cx43 null mutation, we generated mice with selective osteoblast ablation of Cx43, obtained using a Cx43fl allele and a 2.3-kb fragment of the alpha1(I) collagen promoter to drive Cre in osteoblasts (ColCre). Conditionally osteoblast-deleted ColCre;Cx43-/fl mice show no malformations at birth, but develop low peak bone mass and remain osteopenic with age, exhibiting reduced bone formation and defective osteoblast function. By both radiodensitometry and histology, bone mineral content increased rapidly and progressively in adult Cx43+/fl mice after subcutaneous injection of parathyroid hormone (PTH), an effect significantly attenuated in ColCre;Cx43-/fl mice, with Cx43-/fl exhibiting an intermediate response. Attenuation of PTH anabolic action was associated with failure to increase mineral apposition rate in response to PTH in ColCre;Cx43-/fl, despite an increased osteoblast number, suggesting a functional defect in Cx43-deficient bone-forming cells. In conclusion, lack of Cx43 in osteoblasts leads to suboptimal acquisition of peak bone mass, and hinders the bone anabolic effect of PTH. Cx43 represents a potential target for modulation of bone anabolism.

  4. Osteoblast and osteocyte-specific loss of Connexin43 results in delayed bone formation and healing during murine fracture healing.

    PubMed

    Loiselle, Alayna E; Paul, Emmanuel M; Lewis, Gregory S; Donahue, Henry J

    2013-01-01

    Connexin43 (Cx43) plays an important role in osteoblastic differentiation in vitro, and bone formation in vivo. Mice with osteoblast/osteocyte-specific loss of Cx43 display decreased gap junctional intercellular communication (GJIC), bone density, and cortical thickness. To determine the role of Cx43 in fracture healing, a closed femur fracture was induced in Osteocalcin-Cre+; Cx43(flox/flox) (Cx43cKO) and Cre-; Cx43(flox/flox) (WT) mice. We tested the hypothesis that loss of Cx43 results in decreased bone formation and impaired healing following fracture. Here, we show that osteoblast and osteocyte-specific deletion of Cx43 results in decreased bone formation, bone remodeling, and mechanical properties during fracture healing. Cx43cKO mice display decreased bone volume, total volume, and fewer TRAP+ osteoclasts. Furthermore, loss of Cx43 in mature osteoblasts and osteocytes results in a significant decrease in torsional rigidity between 21 and 35 days post-fracture, compared to WT mice. These studies identify a novel role for the gap junction protein Cx43 during fracture healing, suggesting that loss of Cx43 can result in both decreased bone formation and bone resorption. Therefore, enhancing Cx43 expression or GJIC may provide a novel means to enhance bone formation during fracture healing.

  5. Inhibition of GSK-3β rescues the impairments in bone formation and mechanical properties associated with fracture healing in osteoblast selective connexin 43 deficient mice.

    PubMed

    Loiselle, Alayna E; Lloyd, Shane A J; Paul, Emmanuel M; Lewis, Gregory S; Donahue, Henry J

    2013-01-01

    Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.

  6. Connexin43 Hemichannel-Mediated Regulation of Connexin43

    PubMed Central

    Li, Kai; Chi, Yuan; Gao, Kun; Yan, Qiaojing; Matsue, Hiroyuki; Takeda, Masayuki; Kitamura, Masanori; Yao, Jian

    2013-01-01

    Background Many signaling molecules and pathways that regulate gap junctions (GJs) protein expression and function are, in fact, also controlled by GJs. We, therefore, speculated an existence of the GJ channel-mediated self-regulation of GJs. Using a cell culture model in which nonjunctional connexin43 (Cx43) hemichannels were activated by cadmium (Cd2+), we tested this hypothesis. Principal Findings Incubation of Cx43-transfected LLC-PK1 cells with Cd2+ led to an increased expression of Cx43. This effect of Cd2+ was tightly associated with JNK activation. Inhibition of JNK abolished the elevation of Cx43. Further analysis revealed that the changes of JNK and Cx43 were controlled by GSH. Supplement of a membrane-permeable GSH analogue GSH ethyl ester or GSH precursor N-acetyl-cystein abrogated the effects of Cd2+ on JNK activation and Cx43 expression. Indeed, Cd2+ induced extracellular release of GSH. Blockade of Cx43 hemichannels with heptanol or Cx43 mimetic peptide Gap26 to prevent the efflux of GSH significantly attenuated the Cx43-elevating effects of Cd2+. Conclusions Collectively, our results thus indicate that Cd2+-induced upregulation of Cx43 is through activation of nonjunctional Cx43 hemichannels. Our findings thus support the existence of a hemichannel-mediated self-regulation of Cx43 and provide novel insights into the molecular mechanisms of Cx43 expression and function. PMID:23460926

  7. Role of connexin 43 in cardiovascular diseases.

    PubMed

    Michela, Pecoraro; Velia, Verrilli; Aldo, Pinto; Ada, Popolo

    2015-12-01

    Gap junctions (GJs) channels provide the basis for intercellular communication in the cardiovascular system for maintenance of the normal cardiac rhythm, regulation of vascular tone and endothelial function as well as metabolic interchange between the cells. They allow the transfer of small molecules and may enable slow calcium wave spreading, transfer of "death" or of "survival" signals. In the cardiomyocytes the most abundant isoform is Connexin 43 (Cx43). Alterations in Cx43 expression and distribution were observed in myocardium disease; i.e. in hypertrophic cardiomyopathy, heart failure and ischemia. Recent reports suggest the presence of Cx43 in the mitochondria as well, at least in the inner mitochondrial membrane, where it plays a central role in ischemic preconditioning. In this review, the current knowledge on the relationship between the remodeling of cardiac gap junctions and cardiac diseases are summarized.

  8. CONNEXIN 43 PHOSPHORYLATION – STRUCTURAL CHANGES AND BIOLOGICAL EFFECTS

    PubMed Central

    Solan, Joell L.; Lampe, Paul D.

    2009-01-01

    SYNOPSIS Vertebrate gap junctions, composed of proteins from the connexin gene family, play critical roles in embryonic development, coordinated contraction of excitable cells, tissue homeostasis, normal cell growth and differentiation. Phosphorylation of connexin43, the most abundant and ubiquitously expressed connexin, has been implicated in the regulation of gap junctional communication at several stages of the connexin “life cycle” including hemichannel oligomerization, export of the protein to the plasma membrane, hemichannel activity, gap junction assembly, gap junction channel gating and connexin degradation. Consistent with a short (1−5 h) protein half-life, connexin43 phosphorylation is dynamic and changes in response to activation of many different kinases. This review assesses our current understanding of the effects of phosphorylation on connexin43 structure and function that in turn regulate gap junction biology with an emphasis on events occurring in heart and skin. PMID:19309313

  9. Connexin43 mimetic peptide is neuroprotective and improves function following spinal cord injury.

    PubMed

    O'Carroll, Simon J; Gorrie, Catherine A; Velamoor, Sailakshmi; Green, Colin R; Nicholson, Louise F B

    2013-03-01

    Connexin43 (Cx43) is a gap junction protein up-regulated after spinal cord injury and is involved in the on-going spread of secondary tissue damage. To test whether a connexin43 mimetic peptide (Peptide5) reduces inflammation and tissue damage and improves function in an in vivo model of spinal cord injury, rats were subjected to a 10g, 12.5mm weight drop injury at the vertebral level T10 using a MASCIS impactor. Vehicle or connexin43 mimetic peptide was delivered directly to the lesion via intrathecal catheter and osmotic mini-pump for up to 24h after injury. Treatment with Peptide5 led to significant improvements in hindlimb function as assessed using the Basso-Beattie-Bresnahan scale. Peptide5 caused a reduction in Cx43 protein, increased Cx43 phosphorylation and decreased levels of TNF-α and IL-1β as assessed by Western blotting. Immunohistochemistry of tissue sections 5 weeks after injury showed reductions in astrocytosis and activated microglia as well as an increase in motor neuron survival. These results show that administration of a connexin mimetic peptide reduces secondary tissue damage after spinal cord injury by reducing gliosis and cytokine release and indicate the clinical potential for mimetic peptides in the treatment of spinal cord patients. PMID:23403365

  10. Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

    SciTech Connect

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue; Panda, Satya P.

    2011-08-05

    Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

  11. The regulation of runt-related transcription factor 2 by fibroblast growth factor-2 and connexin43 requires the inositol polyphosphate/protein kinase Cδ cascade.

    PubMed

    Niger, Corinne; Luciotti, Maria A; Buo, Atum M; Hebert, Carla; Ma, Vy; Stains, Joseph P

    2013-06-01

    Connexin43 (Cx43) plays a critical role in osteoblast function and bone mass accrual, yet the identity of the second messengers communicated by Cx43 gap junctions, the targets of these second messengers and how they regulate osteoblast function remain largely unknown. We have shown that alterations of Cx43 expression in osteoblasts can impact the responsiveness to fibroblast growth factor-2 (FGF2), by modulating the transcriptional activity of runt-related transcription factor 2 (Runx2). In this study, we examined the contribution of the phospholipase Cγ1/inositol polyphosphate/protein kinase C delta (PKCδ) cascade to the Cx43-dependent transcriptional response of MC3T3 osteoblasts to FGF2. Knockdown of expression and/or inhibition of function of phospholipase Cγ1, inositol polyphosphate multikinase, which generates inositol 1,3,4,5-tetrakisphosphate (InsP₄) and InsP₅, and inositol hexakisphosphate kinase 1/2, which generates inositol pyrophosphates, prevented the ability of Cx43 to potentiate FGF2-induced signaling through Runx2. Conversely, overexpression of phospholipase Cγ1 and inositol hexakisphosphate kinase 1/2 enhanced FGF2 activation of Runx2 and the effect of Cx43 overexpression on this response. Disruption of these pathways blocked the nuclear accumulation of PKCδ and the FGF2-dependent interaction of PKCδ and Runx2, reducing Runx2 transcriptional activity. These data reveal that FGF2-signaling involves the inositol polyphosphate cascade, including inositol hexakisphosphate kinase (IP6K), and demonstrate that IP6K regulates Runx2 and osteoblast gene expression. Additionally, these data implicate the water-soluble inositol polyphosphates as mediators of the Cx43-dependent amplification of the osteoblast response to FGF2, and suggest that these low molecular weight second messengers may be biologically relevant mediators of osteoblast function that are communicated by Cx43-gap junctions.

  12. Fibrosis, Connexin-43, and Conduction Abnormalities in the Brugada Syndrome

    PubMed Central

    Nademanee, Koonlawee; Raju, Hariharan; de Noronha, Sofia V.; Papadakis, Michael; Robinson, Laurence; Rothery, Stephen; Makita, Naomasa; Kowase, Shinya; Boonmee, Nakorn; Vitayakritsirikul, Vorapot; Ratanarapee, Samrerng; Sharma, Sanjay; van der Wal, Allard C.; Christiansen, Michael; Tan, Hanno L.; Wilde, Arthur A.; Nogami, Akihiko; Sheppard, Mary N.; Veerakul, Gumpanart; Behr, Elijah R.

    2015-01-01

    Background The right ventricular outflow tract (RVOT) is acknowledged to be responsible for arrhythmogenesis in Brugada syndrome (BrS), but the pathophysiology remains controversial. Objectives This study assessed the substrate underlying BrS at post-mortem and in vivo, and the role for open thoracotomy ablation. Methods Six whole hearts from male post-mortem cases of unexplained sudden death (mean age 23.2 years) with negative specialist cardiac autopsy and familial BrS were used and matched to 6 homograft control hearts by sex and age (within 3 years) by random risk set sampling. Cardiac autopsy sections from cases and control hearts were stained with picrosirius red for collagen. The RVOT was evaluated in detail, including immunofluorescent stain for connexin-43 (Cx43). Collagen and Cx43 were quantified digitally and compared. An in vivo study was undertaken on 6 consecutive BrS patients (mean age 39.8 years, all men) during epicardial RVOT ablation for arrhythmia via thoracotomy. Abnormal late and fractionated potentials indicative of slowed conduction were identified, and biopsies were taken before ablation. Results Collagen was increased in BrS autopsy cases compared with control hearts (odds ratio [OR]: 1.42; p = 0.026). Fibrosis was greatest in the RVOT (OR: 1.98; p = 0.003) and the epicardium (OR: 2.00; p = 0.001). The Cx43 signal was reduced in BrS RVOT (OR: 0.59; p = 0.001). Autopsy and in vivo RVOT samples identified epicardial and interstitial fibrosis. This was collocated with abnormal potentials in vivo that, when ablated, abolished the type 1 Brugada electrocardiogram without ventricular arrhythmia over 24.6 ± 9.7 months. Conclusions BrS is associated with epicardial surface and interstitial fibrosis and reduced gap junction expression in the RVOT. This collocates to abnormal potentials, and their ablation abolishes the BrS phenotype and life-threatening arrhythmias. BrS is also associated with increased collagen throughout the heart

  13. Bushen-Qiangdu-Zhilv decoction inhibits osteogenic differentiation of rat fibroblasts by regulating connexin 43

    PubMed Central

    ZHOU, YING-YAN; HUANG, RUN-YUE; LIN, JIE-HUA; XU, YONG-YUE; HE, XIAO-HONG; HE, YI-TING

    2016-01-01

    Bushen-Qiangdu-Zhilv (BQZ) decoction is a traditional Chinese medicinal compound widely used for treating ankylosing spondylitis (AS). However, the mechanisms underlying effects of BQZ remain largely unknown. Osteoblast differentiation of fibroblasts plays an important role in heterotopic ossification (HO) of AS, and connexin 43 (Cx43) is crucially involved in the osteoblast differentiation of fibroblasts. The aim of the present study was to evaluate the effects of BQZ on the osteogenic differentiation of fibroblasts by regulating Cx43. Rat fibroblasts were treated with freeze-dried powder of BQZ, in the presence or absence of recombinant human bone morphogenetic protein-2 (rhBMP-2). MTS assays were performed to examine the inhibitory effects of BQZ on fibroblast proliferation. Western blot assays were conducted to detect the protein expression of core-binding factor alpha 1 (Cbfα1), Cx43 and phosphorylated Cx43 (pCx43). BQZ appeared to inhibit fibroblast proliferation in a dose-dependent manner. Furthermore, the expression of Cbfα1 and Cx43/pCx43 was significantly suppressed by BQZ, with or without rhBMP-2 stimulation. Therefore, the present results indicate that BQZ may exert an anti-AS effect by suppressing the osteogenic differentiation of fibroblasts via Cx43 regulation. PMID:27347061

  14. Osteoinductivity of Calcium Phosphate Mediated by Connexin 43

    PubMed Central

    Syed-Picard, Fatima N.; Jayaraman, Thottala; Lam, Raymond S.K.; Beniash, Elia; Sfeir, Charles

    2013-01-01

    Recent reports have alluded to the osteoinductive properties of calcium phosphate, yet the cellular processes behind this are not well understood. To gain insight into the molecular mechanisms of this phenomenon, we have conducted a series of in vitro and in vivo experiments using a scaffoldless three dimensional (3D) dental pulp cell (DPC) construct as a physiologically relevant model. We demonstrate that amorphous calcium phosphate (ACP) alters cellular functions and 3D spatial tissue differentiation patterns by increasing local calcium concentration, which modulates connexin 43 (Cx43)-mediated gap junctions. These observations indicate a chemical mechanism for osteoinductivity of calcium phosphates. These results provide new insights for possible roles of mineral phases in bone formation and remodeling. This study also emphasizes the strong effect of scaffold materials on cellular functions and is expected to advance the design of future tissue engineering materials. PMID:23465492

  15. Alterations in connexin 43 during diabetic cardiomyopathy: competition of tyrosine nitration versus phosphorylation

    PubMed Central

    COOK, Angela C.; SCHANBACHER, Brandon L.; BAUER, John Anthony

    2014-01-01

    Objective Cardiac conduction abnormalities are observed early in the progression of Type I diabetes, but the mechanism(s) involved are undefined. Connexin 43, a critical component of ventricular gap junctions, depends on tyrosine phosphorylation status to modulate channel conductance - alterations in connexin 43 content, distributions, and/or phosphorylation status may be involved in cardiac rhythm disturbances. We tested the hypothesis that cardiac content/distribution of connexin 43 are altered in a rat model of Type I diabetic cardiomyopathy, investigating a mechanistic role for tyrosine. Methods We conducted electrocardiographic analyses during the progression of diabetic cardiomyopathy in rats dosed with streptozotocin (65mg/kg), at 3, 7, and 35 days post-induction of diabetes. Following functional analyses, we conducted immunohistochemical and immunoprecipitation studies to assess alterations in connexin 43. Results We observed significant evidence of ventricular conduction abnormalities (QRS complex, Q-T interval) as early as 7 days post-streptozotocin, persisting throughout the study. Connexin 43 levels were increased 7d post- streptozotocin and remained elevated throughout the study. Connexin 40 content was unchanged relative to controls throughout the study. Changes in Connexin 43 distribution were also observed; connexin 43 staining was dispersed from myocyte short axis junctions. Connexin 43 tyrosine phosphorylation declined during the progression of diabetes, with concurrent increases in tyrosine nitration. Conclusions These data suggest that alterations in connexin 43 content and distribution occur during experimental diabetes and likely contribute to alterations in cardiac function, and that oxidative modification of tyrosine-mediated signaling may play a mechanistic role. PMID:24796789

  16. Short-term pacing in the mouse alters cardiac expression of connexin43

    PubMed Central

    Kontogeorgis, Andrianos; Kaba, Riyaz A; Kang, Eunice; Feig, Jonathan E; Gupta, Pritha P; Ponzio, Marc; Liu, Fangyu; Rindler, Michael J; Wit, Andrew L; Fisher, Edward A; Peters, Nicholas S; Gutstein, David E

    2008-01-01

    Background Cardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time. Results The subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10–15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias. Conclusion Our data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease. PMID:18460209

  17. Role of CAPE on cardiomyocyte protection via connexin 43 regulation under hypoxia

    PubMed Central

    Chen, Chien-Cheng; Kuo, Chan-Yen; Chen, Rong-Fu

    2016-01-01

    Background: Cardiomyocyte under hypoxia cause cell death or damage is associated with heart failure. Gap junction, such as connexin 43 play a role in regulation of heart function under hypoxia. Caffeic acid phenethyl ester (CAPE) has been reported as an active component of propolis, has antioxidative, anti-inflammatory antiproliferative and antineoplastic biological properties. Aims: Connexin 43 appear to have a critical role in heart failure under hypoxia, there has been considerable interest in identifying the candidate component or compound to reduce cell death. Methods: In this study, we used human cardiomyocyte as a cell model to study the role of connexin 43 in hypoxia- incubated human cardiomyocyte in absence or presence of CAPE treatment. Results: Results showed that hypoxia induced connexin 43 expression, but not altered in connexin 40. Interestingly, CAPE attenuates hypoxia-caused connexin 43 down-regulation and cell death or cell growth inhibition. Conclusion: We suggested that reduction of cell death in cardiomyocytes by CAPE is associated with an increase in connexin 43 expression. PMID:27766024

  18. Proteomic and metabolomic responses to connexin43 silencing in primary hepatocyte cultures.

    PubMed

    Vinken, Mathieu; Maes, Michaël; Cavill, Rachel; Valkenborg, Dirk; Ellis, James K; Decrock, Elke; Leybaert, Luc; Staes, An; Gevaert, Kris; Oliveira, André G; Menezes, Gustavo B; Cogliati, Bruno; Dagli, Maria Lúcia Zaidan; Ebbels, Timothy M D; Witters, Erwin; Keun, Hector C; Vanhaecke, Tamara; Rogiers, Vera

    2013-05-01

    Freshly established cultures of primary hepatocytes progressively adopt a foetal-like phenotype and display increased production of connexin43. The latter is a multifaceted cellular entity with variable subcellular locations, including the mitochondrial compartment. Cx43 forms hemichannels and gap junctions that are involved in a plethora of physiological and pathological processes, such as apoptosis. The present study was conducted with the goal of shedding more light onto the role of connexin43 in primary hepatocyte cultures. Connexin43 expression was suppressed by means of RNA interference technology, and the overall outcome of this treatment on the hepatocellular proteome and metabolome was investigated using tandem mass tag-based differential protein profiling and (1)H NMR spectroscopy, respectively. Global protein profiling revealed a number of targets of the connexin43 knock-down procedure, including mitochondrial proteins (heat shock protein 60, glucose-regulated protein 75, thiosulphate sulphurtransferase and adenosine triphosphate synthase) and detoxifying enzymes (glutathione S-transferase μ 2 and cytochrome P450 2C70). At the metabolomic level, connexin43 silencing caused no overt changes, though there was some evidence for a subtle increase in intracellular glycine quantities. Collectively, these data could further substantiate the established existence of a mitochondrial connexin pool and could be reconciled with the previously reported involvement of connexin43 signalling in spontaneously occurring apoptosis in primary hepatocyte cultures.

  19. Inhibition of connexin43 gap junction channels by the endocrine disruptor ioxynil

    SciTech Connect

    Leithe, Edward; Kjenseth, Ane; Bruun, Jarle; Sirnes, Solveig; Rivedal, Edgar

    2010-08-15

    Gap junctions are intercellular plasma membrane domains containing channels that mediate transport of ions, metabolites and small signaling molecules between adjacent cells. Gap junctions play important roles in a variety of cellular processes, including regulation of cell growth and differentiation, maintenance of tissue homeostasis and embryogenesis. The constituents of gap junction channels are a family of trans-membrane proteins called connexins, of which the best-studied is connexin43. Connexin43 functions as a tumor suppressor protein in various tissue types and is frequently dysregulated in human cancers. The pesticide ioxynil has previously been shown to act as an endocrine disrupting chemical and has multiple effects on the thyroid axis. Furthermore, both ioxynil and its derivative ioxynil octanoate have been reported to induce tumors in animal bioassays. However, the molecular mechanisms underlying the possible tumorigenic effects of these compounds are unknown. In the present study we show that ioxynil and ioxynil octanoate are strong inhibitors of connexin43 gap junction channels. Both compounds induced rapid loss of connexin43 gap junctions at the plasma membrane and increased connexin43 degradation. Ioxynil octanoate, but not ioxynil, was found to be a strong activator of ERK1/2. The compounds also had different effects on the phosphorylation status of connexin43. Taken together, the data show that ioxynil and ioxynil octanoate are potent inhibitors of intercellular communication via gap junctions.

  20. Opposing roles of connexin43 in glioma progression.

    PubMed

    Sin, Wun-Chey; Crespin, Sophie; Mesnil, Marc

    2012-08-01

    Despite the tremendous amount of data over the last 40years, lack of gap junctional intercellular communication (GJIC) or altered expression of gap junction proteins is still a lesser known 'hallmark' of cancer. Expression of astrocytic gap junction protein, connexin43 (Cx43), is often reduced in astrocytomas, the most common neoplasia of the central nervous system (CNS) in adults. Supported by a number of evidences, the global decrease of Cx43 expression appears to be advantageous for the growth of glioma cells. Although the mechanisms by which Cx43 regulates the expression levels of proteins involved in cell growth is unclear, there are evidences to suggest that it might be independent of their channel forming properties. In this regard, the carboxyl tail of Cx43 may have the ability to control the translocation of transcription factor regulators into the nucleus. However, this putative tumor suppressor effect of Cx43 is counterbalanced by its capacity to enhance the migration of glioma cells out of the tumor core through mechanisms that seems to implicate its carboxyl tail, possibly by interacting with the actin cytoskeleton. This ambivalence between the tumor suppressor effect and promotion of cell migration may partly be explained by the heterogeneous expression of Cx43 in the glioma core especially at the malignant glioblastoma stage; some tumor cells would be expected to migrate (Cx43 expressing cells) and others to proliferate (non-expressing Cx43 cells). Moreover, the involvement of Cx43 in glioma progression seems to be more complex since, in addition, GJIC may increase their resistance to apoptosis and Cx43 may also affect cell homeostasis in a paracrine fashion via hemichannel action. In conclusion, Cx43 appears to be involved at different levels of the glioma progression by acting on cell growth regulation, promotion of cell migration and resistance to apoptosis. This article is part of a Special Issue entitled: The Communicating junctions, composition

  1. Carbenoxolone induces permeability transition pore opening in rat mitochondria via the translocator protein TSPO and connexin43.

    PubMed

    Azarashvili, Tamara; Baburina, Yulia; Grachev, Dmitry; Krestinina, Olga; Papadopoulos, Vassilios; Lemasters, John J; Odinokova, Irina; Reiser, Georg

    2014-09-15

    Ca(2+)-induced permeability transition pore (mPTP) opening in isolated rat brain mitochondria is promoted through targeting of connexin43. After a threshold Ca(2+) load, mitochondrial membrane potential drops and efflux of accumulated Ca(2+) from the mitochondrial matrix occurs, indicating the mPTP opening. Specific antibodies were used to assess the role of the translocator protein (18kDa; TSPO) and connexin43 in swelling of isolated rat liver and brain mitochondria induced by carbenoxolone and the endogenous TSPO ligand protoporphyrin IX. Mitochondrial membrane potential, Ca(2+) transport and oxygen consumption were determined using selective electrodes. All the parameters were detected simultaneously in a chamber with the selective electrodes. The phosphorylation state of mitochondrial protein targets was assessed. We report that Ca(2+)-induced mitochondrial swelling was strengthened in the presence of both carbenoxolone and protoporphyrin IX. The carbenoxolone- and protoporphyrin IX-accelerated mPTP induction in brain mitochondria was completely prevented by antibodies specific for the mitochondrial translocator protein (TSPO). The anti-TSPO antibodies were more effective than anti-сonnexin43 antibodies. Moreover, carbenoxolone-stimulated phosphorylation of mitochondrial proteins was inhibited by anti-TSPO antibodies. Taken together, the data suggests that, in addition to acting via connexion43, carbenoxolone may exert its effect on mPTP via mitochondrial outer membrane TSPO.

  2. The dual effect of ephaptic coupling on cardiac conduction with heterogeneous expression of connexin 43.

    PubMed

    Wei, Ning; Mori, Yoichiro; Tolkacheva, Elena G

    2016-05-21

    Decreased and heterogeneous expression of connexin 43 (Cx43) are common features in animal heart failure models. Ephpatic coupling, which relies on the presence of junctional cleft space between the ends of adjacent cells, has been suggested to play a more active role in mediating intercellular electrical communication when gap junctions are reduced. To better understand the interplay of Cx43 expression and ephaptic coupling on cardiac conduction during heart failure, we performed numerical simulations on our model when Cx43 expression is reduced and heterogeneous. Under severely reduced Cx43 expression, we identified three new phenomena in the presence of ephaptic coupling: alternating conduction, in which ephaptic and gap junction-mediated mechanisms alternate; instability of planar fronts; and small amplitude action potential (SAP), which has a smaller potential amplitude than the normal action potential. In the presence of heterogeneous Cx43 expression, ephaptic coupling can either prevent or promote conduction block (CB) depending on the Cx43 knockout (Cx43KO) content. When Cx43KO content is relatively high, ephaptic coupling reduces the probabilities of CB. However, ephaptic coupling promotes CB when Cx43KO and wild type cells are mixed in roughly equal proportion, which can be attributed to an increase in current-to-load mismatch.

  3. Oxygen Sensitivity of Placental Trophoblast Connexins 43 and 46: A Role in Preeclampsia?

    PubMed

    Otto, Teresa; Gellhaus, Alexandra; Lüschen, Navina; Scheidler, Jan; Bendix, Ivo; Dunk, Caroline; Wolf, Nadine; Lennartz, Klaus; Köninger, Angela; Schmidt, Markus; Kimmig, Rainer; Fandrey, Joachim; Winterhager, Elke

    2015-12-01

    Several gap junction connexins have been shown to be essential for appropriate placental development and function. It is known that the expression and distribution of connexins change in response to environmental oxygen levels. The placenta develops under various oxygen levels, beginning at a low oxygen tension of approximately 2% and increasing to a tension of 8% after the onset of the uteroplacental circulation. Moreover, it has been shown that during preeclampsia (PE) placentas are subjected to chronic hypoxia. Therefore, we investigated oxygen sensitivity of placental connexins 43 and 46. Using the trophoblast cell line Jar, we demonstrated that the expression of connexin43 increased during acute hypoxia but decreased during chronic hypoxia. Chronic hypoxia resulted in the translocation of connexin43 from the membrane to the cytoplasm and in a reduction in its communication properties. In contrast, the expression of connexin46 was down-regulated during chronic hypoxia and was translocated from perinuclear areas to the cell membrane. Hypoxia-inducible factor (HIF) knockdown showed that the translocation of connexin43 but not that of connexin46 was HIF-2α dependent and was mediated by phosphoinositide 3-kinase. The up-regulation of connexin43 in combination with the down-regulation of connexin46 was confirmed in placental explants cultivated under low oxygen and in placentas with early-onset PE. Taken together, in Jar cells, placental connexins 43 and 46 are regulated during periods of low oxygen in opposite manners. The oxygen sensing of connexins in the trophoblast may play a role in physiological and pathophysiological oxygen conditions and thus may contribute to PE.

  4. Connexin 43 contributes to ectopic orofacial pain following inferior alveolar nerve injury

    PubMed Central

    Shinoda, Masamichi; Honda, Kuniya; Unno, Syumpei; Shimizu, Noriyoshi; Iwata, Koichi

    2016-01-01

    Background Clinically, it is well known that injury of mandibular nerve fiber induces persistent ectopic pain which can spread to a wide area of the orofacial region innervated by the uninjured trigeminal nerve branches. However, the exact mechanism of such persistent ectopic orofacial pain is not still known. The present study was undertaken to determine the role of connexin 43 in the trigeminal ganglion on mechanical hypersensitivity in rat whisker pad skin induced by inferior alveolar nerve injury. Here, we examined changes in orofacial mechanical sensitivity following inferior alveolar nerve injury. Furthermore, changes in connexin 43 expression in the trigeminal ganglion and its localization in the trigeminal ganglion were also examined. In addition, we investigated the functional significance of connexin 43 in relation to mechanical allodynia by using a selective gap junction blocker (Gap27). Results Long-lasting mechanical allodynia in the whisker pad skin and the upper eyelid skin, and activation of satellite glial cells in the trigeminal ganglion, were induced after inferior alveolar nerve injury. Connexin 43 was expressed in the activated satellite glial cells encircling trigeminal ganglion neurons innervating the whisker pad skin, and the connexin 43 protein expression was significantly increased after inferior alveolar nerve injury. Administration of Gap27 in the trigeminal ganglion significantly reduced satellite glial cell activation and mechanical hypersensitivity in the whisker pad skin. Moreover, the marked activation of satellite glial cells encircling trigeminal ganglion neurons innervating the whisker pad skin following inferior alveolar nerve injury implies that the satellite glial cell activation exerts a major influence on the excitability of nociceptive trigeminal ganglion neurons. Conclusions These findings indicate that the propagation of satellite glial cell activation throughout the trigeminal ganglion via gap junctions, which are

  5. Connexin43 contributes to electrotonic conduction across scar tissue in the intact heart

    PubMed Central

    Mahoney, Vanessa M.; Mezzano, Valeria; Mirams, Gary R.; Maass, Karen; Li, Zhen; Cerrone, Marina; Vasquez, Carolina; Bapat, Aneesh; Delmar, Mario; Morley, Gregory E.

    2016-01-01

    Studies have demonstrated non-myocytes, including fibroblasts, can electrically couple to myocytes in culture. However, evidence demonstrating current can passively spread across scar tissue in the intact heart remains elusive. We hypothesize electrotonic conduction occurs across non-myocyte gaps in the heart and is partly mediated by Connexin43 (Cx43). We investigated whether non-myocytes in ventricular scar tissue are electrically connected to surrounding myocardial tissue in wild type and fibroblast-specific protein-1 driven conditional Cx43 knock-out mice (Cx43fsp1KO). Electrical coupling between the scar and uninjured myocardium was demonstrated by injecting current into the myocardium and recording depolarization in the scar through optical mapping. Coupling was significantly reduced in Cx43fsp1KO hearts. Voltage signals were recorded using microelectrodes from control scars but no signals were obtained from Cx43fsp1KO hearts. Recordings showed significantly decreased amplitude, depolarized resting membrane potential, increased duration and reduced upstroke velocity compared to surrounding myocytes, suggesting that the non-excitable cells in the scar closely follow myocyte action potentials. These results were further validated by mathematical simulations. Optical mapping demonstrated that current delivered within the scar could induce activation of the surrounding myocardium. These data demonstrate non-myocytes in the scar are electrically coupled to myocytes, and coupling depends on Cx43 expression. PMID:27244564

  6. Connexin43 contributes to electrotonic conduction across scar tissue in the intact heart

    NASA Astrophysics Data System (ADS)

    Mahoney, Vanessa M.; Mezzano, Valeria; Mirams, Gary R.; Maass, Karen; Li, Zhen; Cerrone, Marina; Vasquez, Carolina; Bapat, Aneesh; Delmar, Mario; Morley, Gregory E.

    2016-05-01

    Studies have demonstrated non-myocytes, including fibroblasts, can electrically couple to myocytes in culture. However, evidence demonstrating current can passively spread across scar tissue in the intact heart remains elusive. We hypothesize electrotonic conduction occurs across non-myocyte gaps in the heart and is partly mediated by Connexin43 (Cx43). We investigated whether non-myocytes in ventricular scar tissue are electrically connected to surrounding myocardial tissue in wild type and fibroblast-specific protein-1 driven conditional Cx43 knock-out mice (Cx43fsp1KO). Electrical coupling between the scar and uninjured myocardium was demonstrated by injecting current into the myocardium and recording depolarization in the scar through optical mapping. Coupling was significantly reduced in Cx43fsp1KO hearts. Voltage signals were recorded using microelectrodes from control scars but no signals were obtained from Cx43fsp1KO hearts. Recordings showed significantly decreased amplitude, depolarized resting membrane potential, increased duration and reduced upstroke velocity compared to surrounding myocytes, suggesting that the non-excitable cells in the scar closely follow myocyte action potentials. These results were further validated by mathematical simulations. Optical mapping demonstrated that current delivered within the scar could induce activation of the surrounding myocardium. These data demonstrate non-myocytes in the scar are electrically coupled to myocytes, and coupling depends on Cx43 expression.

  7. Neuroprotection in the treatment of glaucoma--A focus on connexin43 gap junction channel blockers.

    PubMed

    Chen, Ying-Shan; Green, Colin R; Danesh-Meyer, Helen V; Rupenthal, Ilva D

    2015-09-01

    Glaucoma is a form of optic neuropathy and a common cause of blindness, affecting over 60 million people worldwide with an expected rise to 80 million by 2020. Successful treatment is challenging due to the various causes of glaucoma, undetectable symptoms at an early stage and inefficient delivery of drugs to the back of the eye. Conventional glaucoma treatments focus on the reduction of elevated intraocular pressure (IOP) using topical eye drops. However, their efficacy is limited to patients who suffer from high IOP glaucoma and do not address the underlying susceptibility of retinal ganglion cells (RGC) to degeneration. Glaucoma is known as a neurodegenerative disease which starts with RGC death and eventually results in damage of the optic nerve. Neuroprotective strategies therefore offer a novel treatment option for glaucoma by not only preventing neuronal loss but also disease progression. This review firstly gives an overview of the pathophysiology of glaucoma as well as current treatment options including conventional and novel delivery strategies. It then summarizes the rational for neuroprotection as a novel therapy for glaucomatous neuropathies and reviews current potential neuroprotective strategies to preserve RGC, with a focus on connexin43 (Cx43) gap junction channel blockers.

  8. Electrical remodeling contributes to complex tachyarrhythmias in connexin43-deficient mouse hearts

    PubMed Central

    Danik, Stephan B.; Rosner, Gregg; Lader, Joshua; Gutstein, David E.; Fishman, Glenn I.; Morley, Gregory E.

    2009-01-01

    Loss of connexin43 (Cx43) gap junction channels in the heart results in a marked increase in the incidence of spontaneous and inducible polymorphic ventricular tachyarrhythmias (PVTs). The mechanisms resulting in this phenotype remain unclear. We hypothesized that uncoupling promotes regional ion channel remodeling, thereby increasing electrical heterogeneity and facilitating the development of PVT. In isolated-perfused control hearts, programmed electrical stimulation elicited infrequent monomorphic ventricular tachyarrhythmias (MVT), and dominant frequencies (DFs) during MVT were similar in the right ventricle (RV) and left ventricle (LV). Moreover, conduction properties, action potential durations (APDs), and repolarizing current densities were similar in RV and LV myocytes. In contrast, PVT was common in Cx43 conditional knockout (OCKO) hearts, and arrhythmias were characterized by significantly higher DFs in the RV compared to the LV. APDs in OCKO myocytes were significantly shorter than those from chamber-matched controls, with RV OCKO myocytes being most affected. APD shortening was associated with higher levels of sustained current in myocytes from both chambers as well as higher levels of the inward rectifier current only in RV myocytes. Thus, alterations in cell-cell coupling lead to regional changes in potassium current expression, which in this case facilitates the development of reentrant arrhythmias. We propose a new mechanistic link between electrical uncoupling and ion channel remodeling. These findings may be relevant not only in cardiac tissue but also to other organ systems where gap junction remodeling is known to occur. PMID:17984180

  9. Electrical remodeling contributes to complex tachyarrhythmias in connexin43-deficient mouse hearts.

    PubMed

    Danik, Stephan B; Rosner, Gregg; Lader, Joshua; Gutstein, David E; Fishman, Glenn I; Morley, Gregory E

    2008-04-01

    Loss of connexin43 (Cx43) gap junction channels in the heart results in a marked increase in the incidence of spontaneous and inducible polymorphic ventricular tachyarrhythmias (PVTs). The mechanisms resulting in this phenotype remain unclear. We hypothesized that uncoupling promotes regional ion channel remodeling, thereby increasing electrical heterogeneity and facilitating the development of PVT. In isolated-perfused control hearts, programmed electrical stimulation elicited infrequent monomorphic ventricular tachyarrhythmias (MVT), and dominant frequencies (DFs) during MVT were similar in the right ventricle (RV) and left ventricle (LV). Moreover, conduction properties, action potential durations (APDs), and repolarizing current densities were similar in RV and LV myocytes. In contrast, PVT was common in Cx43 conditional knockout (OCKO) hearts, and arrhythmias were characterized by significantly higher DFs in the RV compared to the LV. APDs in OCKO myocytes were significantly shorter than those from chamber-matched controls, with RV OCKO myocytes being most affected. APD shortening was associated with higher levels of sustained current in myocytes from both chambers as well as higher levels of the inward rectifier current only in RV myocytes. Thus, alterations in cell-cell coupling lead to regional changes in potassium current expression, which in this case facilitates the development of reentrant arrhythmias. We propose a new mechanistic link between electrical uncoupling and ion channel remodeling. These findings may be relevant not only in cardiac tissue but also to other organ systems where gap junction remodeling is known to occur. PMID:17984180

  10. Role of Akt and Ca2+ on cell permeabilization via connexin43 hemichannels induced by metabolic inhibition.

    PubMed

    Salas, Daniela; Puebla, Carlos; Lampe, Paul D; Lavandero, Sergio; Sáez, Juan C

    2015-07-01

    Connexin hemichannels are regulated under physiological and pathological conditions. Metabolic inhibition, a model of ischemia, promotes surface hemichannel activation associated, in part, with increased surface hemichannel levels, but little is known about its underlying mechanism. Here, we investigated the role of Akt on the connexin43 hemichannel's response induced by metabolic inhibition. In HeLa cells stably transfected with rat connexin43 fused to EGFP (HeLa43 cells), metabolic inhibition induced a transient Akt activation necessary to increase the amount of surface connexin43. The increase in levels of surface connexin43 was also found to depend on an intracellular Ca2+ signal increase that was partially mediated by Akt activation. However, the metabolic inhibition-induced Akt activation was not significantly affected by intracellular Ca2+ chelation. The Akt-dependent increase in connexin43 hemichannel activity in HeLa43 cells also occurred after oxygen-glucose deprivation, another ischemia-like condition, and in cultured cortical astrocytes (endogenous connexin43 expression system) under metabolic inhibition. Since opening of hemichannels has been shown to accelerate cell death, inhibition of Akt-dependent phosphorylation of connexin43 hemichannels could reduce cell death induced by ischemia/reperfusion.

  11. Role of Akt and Ca2+ on cell permeabilization via connexin43 hemichannels induced by metabolic inhibition.

    PubMed

    Salas, Daniela; Puebla, Carlos; Lampe, Paul D; Lavandero, Sergio; Sáez, Juan C

    2015-07-01

    Connexin hemichannels are regulated under physiological and pathological conditions. Metabolic inhibition, a model of ischemia, promotes surface hemichannel activation associated, in part, with increased surface hemichannel levels, but little is known about its underlying mechanism. Here, we investigated the role of Akt on the connexin43 hemichannel's response induced by metabolic inhibition. In HeLa cells stably transfected with rat connexin43 fused to EGFP (HeLa43 cells), metabolic inhibition induced a transient Akt activation necessary to increase the amount of surface connexin43. The increase in levels of surface connexin43 was also found to depend on an intracellular Ca2+ signal increase that was partially mediated by Akt activation. However, the metabolic inhibition-induced Akt activation was not significantly affected by intracellular Ca2+ chelation. The Akt-dependent increase in connexin43 hemichannel activity in HeLa43 cells also occurred after oxygen-glucose deprivation, another ischemia-like condition, and in cultured cortical astrocytes (endogenous connexin43 expression system) under metabolic inhibition. Since opening of hemichannels has been shown to accelerate cell death, inhibition of Akt-dependent phosphorylation of connexin43 hemichannels could reduce cell death induced by ischemia/reperfusion. PMID:25779082

  12. Chronic psychotropic drug treatment causes differential expression of connexin 43 and GFAP in frontal cortex of rats.

    PubMed

    Fatemi, S Hossein; Folsom, Timothy D; Reutiman, Teri J; Pandian, Twinkle; Braun, Natalie N; Haug, Kari

    2008-09-01

    Astrocytic markers glial fibrillary acidic protein (GFAP) and connexin 43 (CX43) are known to have altered expression in brains of subjects with psychiatric disorders including autism and major depression. The current study investigated whether GFAP and CX43 expressions are affected by several commonly used psychotropic medications (clozapine, fluoxetine, haloperidol, lithium, olanzapine, and valproic acid). Using SDS-PAGE and western blotting technique, we observed that CX43 protein expression in prefrontal cortex was significantly increased following chronic treatment with fluoxetine and clozapine, while it was significantly decreased by haloperidol and lithium. GFAP protein expression was significantly decreased following chronic treatment with clozapine and valproic acid. These results suggest that astroglial markers GFAP and CX43 could be potential targets for therapeutic intervention.

  13. Nonlinear behaviour of conduction and block in cardiac tissue with heterogeneous expression of connexin 43.

    PubMed

    Prudat, Yann; Kucera, Jan P

    2014-11-01

    Altered gap junctional coupling potentiates slow conduction and arrhythmias. To better understand how heterogeneous connexin expression affects conduction at the cellular scale, we investigated conduction in tissue consisting of two cardiomyocyte populations expressing different connexin levels. Conduction was mapped using microelectrode arrays in cultured strands of foetal murine ventricular myocytes with predefined contents of connexin 43 knockout (Cx43KO) cells. Corresponding computer simulations were run in randomly generated two-dimensional tissues mimicking the cellular architecture of the strands. In the cultures, the relationship between conduction velocity (CV) and Cx43KO cell content was nonlinear. CV first decreased significantly when Cx43KO content was increased from 0 to 50%. When the Cx43KO content was ≥60%, CV became comparable to that in 100% Cx43KO strands. Co-culturing Cx43KO and wild-type cells also resulted in significantly more heterogeneous conduction patterns and in frequent conduction blocks. The simulations replicated this behaviour of conduction. For Cx43KO contents of 10-50%, conduction was slowed due to wavefront meandering between Cx43KO cells. For Cx43KO contents ≥60%, clusters of remaining wild-type cells acted as electrical loads that impaired conduction. For Cx43KO contents of 40-60%, conduction exhibited fractal characteristics, was prone to block, and was more sensitive to changes in ion currents compared to homogeneous tissue. In conclusion, conduction velocity and stability behave in a nonlinear manner when cardiomyocytes expressing different connexin amounts are combined. This behaviour results from heterogeneous current-to-load relationships at the cellular level. Such behaviour is likely to be arrhythmogenic in various clinical contexts in which gap junctional coupling is heterogeneous.

  14. Downregulation of connexin43 by microRNA-130a in cardiomyocytes results in cardiac arrhythmias.

    PubMed

    Osbourne, Appledene; Calway, Tyler; Broman, Michael; McSharry, Saoirse; Earley, Judy; Kim, Gene H

    2014-09-01

    MicroRNAs (miRNAs) are now recognized as critical regulators of diverse physiological and pathological processes; however, studies of miRNAs and arrhythmogenesis remain sparse. Connexin43 (Cx43), a major cardiac gap junction protein, has elicited great interest in its role in arrhythmias. Additionally, Cx43 was a potential target for miR-130a as predicted by several computational algorithms. This study investigates the effect of miR-130a overexpression in the adult heart and its effect on cardiac rhythm. Using a cardiac-specific inducible system, transgenic mice demonstrated both atrial and ventricular arrhythmias. We performed ventricular-programmed electrical stimulation and found that the αMHC-miR130a mice developed sustained ventricular tachycardia beginning 6weeks after overexpression. Western blot analysis demonstrated a steady decline in Cx43 after 2weeks of overexpression with over a 90% reduction in Cx43 levels by 10weeks. Immunofluorescent staining confirmed a near complete loss of Cx43 throughout the heart. To validate Cx43 as a direct target of miR-130a, we performed in vitro target assays in 3T3 fibroblasts and HL-1 cardiomyocytes, both known to endogenously express miR-130a. Using a luciferase reporter fused to the 3'UTR of Cx43, we found a 52.9% reduction in luciferase activity in 3T3 cells (p<0.0001) and a 47.6% reduction in HL-1 cells (p=0.0056) compared to controls. Addition of an antisense miR-130a inhibitor resulted in a loss of inhibitory activity of the Cx43 3'UTR reporter. We have identified an unappreciated role for miR-130a as a direct regulator of Cx43. Overexpression of miR-130a may contribute importantly to gap junction remodeling and to the pathogenesis of atrial and ventricular arrhythmias.

  15. Functionally Active Gap Junctions between Connexin 43-Positive Mesenchymal Stem Cells and Glioma Cells.

    PubMed

    Gabashvili, A N; Baklaushev, V P; Grinenko, N F; Levinskii, A B; Mel'nikov, P A; Cherepanov, S A; Chekhonin, V P

    2015-05-01

    The formation of functional gap junctions between mesenchymal stem cells and cells of low-grade rat glioma C6 cells was studied in in vitro experiments. Immunocytochemical analysis with antibodies to connexin 43 extracellular loop 2 showed that mesenchymal stem cells as well as C6 glioma cells express the main astroglial gap junction protein connexin 43. Analysis of migration activity showed that mesenchymal stem cells actively migrate towards C6 glioma cells. During co-culturing, mesenchymal stem cells and glioma C6 form functionally active gap junctions mediating the transport of cytoplasmic dye from glioma cells to mesenchymal stem cells in the opposite direction. Fluorometry showed that the intensity of transport of low-molecular substances through heterologous gap junctions between mesenchymal stem cells and glioma cells is similar to that through homologous gap junctions between glioma cells. This phenomenon can be used for the development of new methods of cell therapy of high-grade gliomas.

  16. Myocardial connexin-43 and N-Cadherin decrease during vanadium inhalation.

    PubMed

    Fortoul, Teresa I; Soto-Mota, Adrian; Rojas-Lemus, Marcela; Rodriguez-Lara, Vianey; Gonzalez-Villalva, Adriana; Montaño, Luis F; Paez, Araceli; Colin-Barenque, Laura; López-Valdez, Nelly; Cano-Gutiérrez, Gumaro; Bizarro-Nevares, Patricia; Ustarroz-Cano, Martha

    2016-04-01

    Particulate matter air pollution has considerably increased during the last decades; vanadium is a transition element adhered to this particulate matter, and the combustion of fossil fuels is the main source in the atmosphere. It has been reported that air pollution and specifically vanadium exposure increases the probability of suffering arrhythmias; however the biological mechanism of such a relationship remains unknown. It has been established that a diminished presence of N-Cadherin alters the Connexin-43 arrangement, and the consequent altered presence of these proteins predisposes to ventricular heart rate problems. We analyzed myocardial histology and the expression of N-Cadherin and Connexin-43 by immunohistochemistry in mouse that inhaled vanadium. Our results showed a significant and progressive reduction in both N-Cadherin and Connexin-43, as well as the presence of meganucleus; myofibrils disruption, and clumping in the exposed groups were also observed. Our findings add more information about a possible explanation for the arrythmogenic effect observed in dwellers of cities with high particulate matter atmospheric pollution. PMID:26568576

  17. Testicular connexin 43, a precocious molecular target for the effect of environmental toxicants on male fertility

    PubMed Central

    Gilleron, Jérôme; Carette, Diane; Segretain, Dominique

    2011-01-01

    Many recent epidemiological, clinical and experimental findings support the hypothesis that environmental toxicants are responsible for the increasing male reproductive disorders (congenital malformations, declining sperm counts and testicular cancer) over the past 20 years. It has also been reported that exposure to these toxicants, during critical periods of development (fetal and neonatal), represents a more considerable risk for animals and humans than exposure during adulthood. However, the molecular targets for these chemicals have not been clearly identified. Recent studies showed that a family of transmembranous proteins, named connexins, regulates numerous physiological processes involved in testicular development and function, such as Sertoli and germ cell proliferation, differentiation, germ cell migration and apoptosis. In the testis, knockout strategy revealed that connexin 43, the predominant connexin in this organ, is essential for spermatogenesis. In addition, there is evidence that many environmental toxicants could alter testicular connexin 43 by dysregulation of numerous mechanisms controlling its function. In the present work, we propose first to give an overview of connexin expression and intercellular gap junction coupling in the developing fetal and neonatal testes. Second, we underline the impact of maternally chemical exposure on connexin 43 expression in the perinatal developing testis. Lastly, we attempt to link this precocious effect to male offspring fertility. PMID:22332114

  18. Transfection of C6 Glioma Cells with Connexin 43 cDNA: Analysis of Expression, Intercellular Coupling, and Cell Proliferation

    NASA Astrophysics Data System (ADS)

    Zhu, D.; Caveney, S.; Kidder, G. M.; Naus, C. C. G.

    1991-03-01

    C6 glioma cells express low levels of the gap junction protein connexin 43 and its mRNA and display very weak dye coupling. When implanted into the rat cerebrum, these cells quickly give rise to a large glioma. To investigate the role of gap junctions in the tumor characteristics of these cells, we have used Lipofectin-mediated transfection to introduce a full-length cDNA encoding connexin 43. Several transfected clones were obtained that exhibited various amounts of connexin 43 mRNA transcribed from the inserted cDNA. Immunocytochemical analysis revealed an increase in the amount of connexin 43 immunoreactivity in the transfected cells, being localized at areas of intercellular contact as well as in the cytoplasm. The level of dye coupling was also assessed and found to correlate with the amount of connexin 43 mRNA. When cell proliferation was followed over several days, cells expressing the transfected cDNA grew more slowly than nontransfected cells. These transfected cells will be useful in examining the role of gap junctions in tumorigenesis.

  19. Effect of Beta-Carotene on Oxidative Stress and Expression of Cardiac Connexin 43

    PubMed Central

    Novo, Rosangela; Azevedo, Paula S.; Minicucci, Marcos F.; Zornoff, Leonardo A. M.; Paiva, Sergio A. R.

    2013-01-01

    Background Intervention studies have shown an increased mortality in patients who received beta-carotene. However, the mechanisms involved in this phenomenon are still unknown. Objective Evaluate the influence of beta-carotene on oxidative stress and the expression of connexin 43 in rat hearts. Methods Wistar rats, weighing approximately 100 g, were allocated in two groups: Control Group (n = 30), that received the diet routinely used in our laboratory, and Beta-Carotene Group (n = 28), which received beta-carotene (in crystal form, added and mixed to the diet) at a dose of 500 mg of beta carotene/kg of diet. The animals received the treatment until they reached 200-250g, when they were sacrificed. Samples of blood, liver and heart were collected to perform Western blotting and immunohistochemistry for connexin 43; morphometric studies, dosages of beta carotene by high performance liquid chromatography as well as reduced glutathione, oxidized glutathione and lipids hydroperoxides were performed by biochemical analysis. Results Beta-carotene was detected only in the liver of Beta-Carotene Group animals (288 ± 94.7 μg/kg). Levels of reduced/ oxidized glutathione were higher in the liver and heart of Beta-Carotene Group animals (liver - Control Group: 42.60 ± 1.62; liver - Beta-Carotene Group: 57.40 ± 5.90; p = 0.04; heart: - Control Group: 117.40 ± 1.01; heart - Beta-Carotene Group: 121.81 ± 1.32 nmol/mg protein; p = 0.03). The content of total connexin 43 was larger in Beta-Carotene Group. Conclusion Beta-carotene demonstrated a positive effect, characterized by the increase of intercellular communication and improvement of anti-oxidizing defense system. In this model, mechanism does not explain the increased mortality rate observed with the beta-carotene supplementation in clinical studies. PMID:23917457

  20. Use of antibodies in the analysis of connexin 43 turnover and phosphorylation.

    PubMed

    Hertzberg, E L; Sáez, J C; Corpina, R A; Roy, C; Kessler, J A

    2000-02-01

    A series of antipeptide antibodies designed to recognize specific sequences of the gap junction protein connexin 43 (Cx43) were developed and characterized immunochemically and immunohistologically. These antibodies bound to gap junctions and, on Western blots, to 43-kDa (often resolved as a doublet) and 41-kDa proteins in samples from heart, leptomeningeal cells, and brain. Relatively little of the 41-kDa protein was detectable in heart homogenates. Cultured rat leptomeningeal cells expressed high levels of the gap junction protein Cx43 and were used to analyze its turnover and phosphorylation. Pulse-chase experiments in leptomeningeal cells with [(35)S]methionine indicated that the 41-kDa form of connexin 43 was the first immunoprecipitable translation product. Radiolabel subsequently appeared in the lower band of the doublet at 43 kDa, followed by a shift into the higher band and turnover of the protein with a t(1/2) of 2.7 h. Pulse-chase labeling with [(32)P]P(i) indicated that phosphorylation of connexin 43 was limited to the 43-kDa protein, with a t(1/2) of 1.7 h. Treatment with alkaline phosphatase shifted the apparent molecular mass of the 43-kDa protein doublet such that it comigrated with the 41-kDa form. Hence, the 43-kDa protein observed on Western blots of both leptomeningeal cells and heart arises by phosphorylation of the 41 kDa precursor. Phosphorylation of serine residues accounts for most, if not all, of Cx43 phosphorylation in this system.

  1. Astroglial connexin43 contributes to neuronal suffering in a mouse model of Alzheimer's disease.

    PubMed

    Yi, C; Mei, X; Ezan, P; Mato, S; Matias, I; Giaume, C; Koulakoff, A

    2016-10-01

    In Alzheimer's disease (AD), astrocyte properties are modified but their involvement in this pathology is only beginning to be appreciated. The expression of connexins, proteins forming gap junction channels and hemichannels, is increased in astrocytes contacting amyloid plaques in brains of AD patients and APP/PS1 mice. The consequences on their channel functions was investigated in a murine model of familial AD, the APPswe/PS1dE9 mice. Whereas gap junctional communication was not affected, we revealed that hemichannels were activated in astrocytes of acute hippocampal slices containing Aβ plaques. Such hemichannel activity was detected in all astrocytes, whatever their distance from amyloid plaques, but with an enhanced activity in the reactive astrocytes contacting amyloid plaques. Connexin43 was the main hemichannel contributor, however, a minor pannexin1 component was also identified in the subpopulation of reactive astrocytes in direct contact with plaques. Distinct regulatory pathways are involved in connexin and pannexin hemichannel activation. Inflammation triggered pannexin hemichannel activity, whereas connexin43 hemichannels were activated by the increase in resting calcium level of astrocytes. Importantly, hemichannel activation led to the release of ATP and glutamate that contributed to maintain a high calcium level in astrocytes placing them in the center of a vicious circle. The astroglial targeted connexin43 gene knocking-out in APPswe/PS1dE9 mice allowed to diminish gliotransmitter release and to alleviate neuronal damages, reducing oxidative stress and neuritic dystrophies in hippocampal neurons associated to plaques. Altogether, these data highlight the importance of astroglial hemichannels in AD and suggest that blocking astroglial hemichannel activity in astrocytes could represent an alternative therapeutic strategy in AD.

  2. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    SciTech Connect

    Carette, Diane; Perrard, Marie-Hélène; Prisant, Nadia; Gilleron, Jérome; Pointis, Georges; Segretain, Dominique; Durand, Philippe

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ► Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ► Use of cultured seminiferous tubules in bicameral chambers ► Low concentrations of Cr(VI) (10 μg/l) altered the trans-epithelial resistance. ► Cr(VI) did not alter claudin-11 and N-cadherin. ► Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  3. Altered connexin 43 expression underlies age dependent decrease of Treg cell suppressor function in NOD mice

    PubMed Central

    Kuczma, Michal; Wang, Cong-Yi; Ignatowicz, Leszek; Gourdie, Robert; Kraj, Piotr

    2015-01-01

    Type I diabetes (T1D) is one of the most extensively studied autoimmune diseases but the cellular and molecular mechanisms leading to T cell-mediated destruction of insulin-producing β-cells are still not well understood. Here we show that Treg cells in NOD mice undergo age-dependent loss of suppressor functions exacerbated by the decreased ability of activated effector T cells to upregulate Foxp3 and generate Treg cells in the peripheral organs. This age-dependent loss is associated with reduced intercellular communication mediated by gap junctions, which is caused by impaired upregulation and decreased expression of connexin 43. Regulatory functions can be corrected, even in T cells isolated from aged, diabetic mice, by a synergistic activity of retinoic acid, TGF-β, and IL-2, which enhance connexin 43 and Foxp3 expression in Treg cells and restore the ability of conventional CD4+ T cells to upregulate Foxp3 and generate peripherally derived Treg cells. Moreover, we demonstrate that suppression mediated by Treg cells from diabetic mice is enhanced by a novel reagent, which facilitates gap junction aggregation. In summary, our report identifies gap junction-mediated intercellular communication as an important component of the Treg cell suppression mechanism compromised in NOD mice and suggests how Treg mediated immune regulation can be improved. PMID:25911751

  4. Identification of miR-200a as a novel suppressor of connexin 43 in breast cancer cells.

    PubMed

    Ming, Jia; Zhou, Yan; Du, Junze; Fan, Shenghao; Pan, Beibei; Wang, Yinhuan; Fan, Lingjun; Jiang, Jun

    2015-08-17

    Both miRNAs (miRs) and connexin 43 (Cx43) were important regulators of the metastasis of breast cancer, whereas the miRs regulating Cx43 expression in breast cancer cells were still obscure. In the present study, we scanned and found miR-1, miR-206, miR-200a, miR-381, miR-23a/b and miR-186 were functional suppressors of human Cx43 mRNA and protein expression. Specially, we demonstrated that only miR-200a could directly target the 3'-untranslated region (3'-UTR) of human Cx43 gene. Functionally, overexpression of Cx43 in MCF cells potentiated the migration activity, whereas additional miR-200a treatment notably prevented this effect. Finally, we demonstrated that decreased levels of miR-200a and elevated expression of Cx43 in the metastatic breast cancer tissues compared with the primary ones. Thus, we are the first to identify miR-200a as a novel and direct suppressor of human Cx43, indicating that miR200a/Cx43 axis might be a useful diagnostic and therapeutic target of metastatic breast cancer.

  5. Connexin43 knockdown in bone marrow‑derived dendritic cells by small interfering RNA leads to a diminished T-cell stimulation.

    PubMed

    Yu, Fuling; Yan, Hui; Nie, Wencheng; Zhu, Jianhua

    2016-01-01

    Dendritic cells, the most powerful type of antigen‑presenting cells, have the unique ability to induce primary immune responses. Connexin43 expression is upregulated to increase gap junctions when immune cells are exposed to inflammatory factors. The present study applied small‑interfering RNA (siRNA) to decrease connexin43 expression. The results showed that silencing of connexin43 using siRNA resulted in arrest of bone marrow‑derived dendritic cell (BM‑DC) maturation as evidenced by reduced expression of major histocompatibility complex II, CD40, CD80 and CD86. Functionally, connexin43‑silenced BM‑DC showed a markedly decreased capability to induce T-cell stimulation. In conclusion, the present study demonstrated that antigens present on BM‑DCs can be suppressed by connexin43 knockdown in BM‑DCs. The present study therefore presented an effective method to modulate the immunology of BM‑DCs.

  6. Transforming growth factor-β1 up-regulates connexin43 expression in human granulosa cells

    PubMed Central

    Chen, Yu-Ching; Chang, Hsun-Ming; Cheng, Jung-Chien; Tsai, Horng-Der; Wu, Cheng-Hsuan; Leung, Peter C.K.

    2015-01-01

    STUDY QUESTION Does transforming growth factor-β1 (TGF-β1) up-regulate connexin43 (Cx43) to promote cell–cell communication in human granulosa cells? SUMMARY ANSWER TGF-β1 up-regulates Cx43 and increases gap junction intercellular communication activities (GJIC) in human granulosa cells, and this effect occurs via the activin receptor-like kinase (ALK)5-mediated Sma- and Mad-related protein (SMAD)2/3-SMAD4-dependent pathway. WHAT IS KNOWN ALREADY TGF-β1 and its receptors are expressed in human granulosa cells, and follicular fluid contains TGF-β1 protein. In human granulosa cells, Cx43 gap junctions play an important role in the development of follicles and oocytes. STUDY DESIGN, SIZE, DURATION This is an experimental study which was performed over a 1-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing IVF in an academic research center were used as the study models. Cx43 mRNA and protein expression levels were examined after exposure of SVOG cells to recombinant human TGF-β1. An activin/TGF-β type I receptor inhibitor, SB431542, and small interfering RNAs targeting ALK4, ALK5, SMAD2, SMAD3 and SMAD4 were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance. MAIN RESULTS AND THE ROLE OF CHANCE TGF-β1 treatment increased phosphorylation of SMAD2/3 (P < 0.0001) and up-regulated Cx43 mRNA and protein levels (P < 0.001) in SVOG cells and these stimulatory effects were abolished by the TGF-β type I receptor inhibitor SB431542. In addition, the up-regulatory effect of TGF-β1 on Cx43 expression (mRNA and protein) was confirmed in primary

  7. Involvement of connexin43 in the infrasonic noise-induced glutamate release by cultured astrocytes.

    PubMed

    Jiang, Shan; Wang, Yong-Qiang; Xu, Cheng-Feng; Li, Ya-Na; Guo, Rong; Li, Ling

    2014-05-01

    Infrasonic noise/infrasound is a type of environmental noise that threatens public health as a nonspecific biological stressor. Glutamate-related excitotoxicity is thought to be responsible for infrasound-induced impairment of learning and memory. In addition to neurons, astrocytes are also capable of releasing glutamate. In the present study, to identify the effect of infrasound on astroglial glutamate release, cultured astrocytes were exposed to infrasound at 16 Hz, 130 dB for different times. We found that infrasound exposure caused a significant increase in glutamate levels in the extracellular fluid. Moreover, blocking the connexin43 (Cx43) hemichannel or gap junction, decreasing the probability of Cx43 being open or inhibiting of Cx43 expression blocked this increase. The results suggest that glutamate release by Cx43 hemichannels/gap junctions is involved in the response of cultured astrocytes to infrasound. PMID:24634254

  8. Expression of CONNEXIN43 is highly sensitive to ionizing radiation and other environmental stresses.

    PubMed

    Azzam, Edouard I; de Toledo, Sonia M; Little, John B

    2003-11-01

    To gain a greater understanding of the mechanisms underlying the cellular responses to low-dose ionizing radiation, gene expression profiles were examined by microarray analysis of cDNA from confluent human diploid fibroblast cultures exposed to very low fluences of alpha-particles. The data, supported by Northern and Western analyses, indicate that radiation induces a significant up-regulation of CONNEXIN43 expression. This phenomenon was observed in a variety of irradiated cell types. These findings are consistent with our previous observations that connexin43 (cx43)-mediated gap-junction intercellular communication is involved in the bystander response observed in cell cultures exposed to fluences of alpha-particles by which only a very small fraction of the cell nuclei is traversed by a particle track (E. I. Azzam et al., Proc. Natl. Acad. Sci. USA, 98: 473-478, 2001). Increased mRNA levels in cells from irradiated cultures correlated with increased cx43 protein levels by approximately 4 h after irradiation. The induction of cx43 was observed by mean alpha-particle doses as low as 0.16 cGy, and also in cells exposed to gamma-rays, t-butyl hydroperoxide, and hyperthermia. Exposure to these stresses also resulted in post-translational modification of cx43; increased phosphorylation and hyperphosphorylation of the protein was observed. Up-regulation of cx43 expression in ionizing radiation exposed cells correlated with functional communication through gap junctions, as evidenced by dye transfer from irradiated to nonirradiated cells. In contrast, the response after UV radiation varied and was cell type-dependent. Overall, these data suggest a critical role for genes involved in intercellular communication in mediating the cellular responses to a variety of stresses. PMID:14612506

  9. Immunohistochemical evaluation of cardiac connexin43 in rats exposed to low-frequency noise

    PubMed Central

    Antunes, Eduardo; Borrecho, Gonçalo; Oliveira, Pedro; Brito, José; Águas, Artur; Martins dos Santos, José

    2013-01-01

    Introduction: Low-frequency noise (LFN) leads to an abnormal proliferation of collagen and development of tissue fibrosis. It has been shown that myocardial fibrosis in association with gap junction remodeling occurs in several cardiac diseases and can be implicated in the development of ventricular tachyarrhythmias. We previously reported a strong development of myocardial fibrosis induced by LFN in rats but it is not known whether LFN induces any modification on cardiac connexin43 (Cx43). Objectives: The aim of this study was to evaluate modifications on cardiac Cx43 induced by LFN in Wistar rats. Methods: Two groups of rats were considered: A LFN-exposed group with 10 rats submitted continuously to LFN during 3 months and a control group with 8 rats. The hearts were sectioned from the ventricular apex to the atria and the mid-ventricular fragment was selected. The immunohistochemical evaluation of Cx43 was performed using the polyclonal antibody connexin-43m diluted 1:1000 overnight at 4°C. Quantifications of Cx43 and muscle were performed with the image J software and the ratio Cx43/muscle was analyzed in the left ventricle, interventricular septum and right ventricle. Results: The ratio Cx43/muscle was significantly reduced in LFN-exposed rats (p=0.001). The mean value decreased 46.2%, 22.2% and 55.6% respectively in the left ventricle (p=0.008), interventricular septum (p=0.301) and right ventricle (p=0.004). Conclusions: LFN induces modifications on cardiac Cx43 in rats. The Cx43 reduction observed in our study suggests that LFN may induce an arrhythmogenic substrate and opens a new investigational area concerning the effects of LFN on the heart. PMID:24040453

  10. Gap junction signalling mediated through connexin-43 is required for chick limb development.

    PubMed

    Makarenkova, H; Patel, K

    1999-03-15

    During chick limb development the gap junction protein Connexin-43 (Cx43) is expressed in discrete spatially restricted domains in the apical ectodermal ridge (AER) and mesenchyme of the zone of polarising activity. Antisense oligonucleotides (ODNs) were used to investigate the role of Connexin-43 (Cx43) in the development of the chick limb bud. We have used unmodified ODNs in Pluronic F-127 gel, which is liquid at low temperature but sets at room temperature and so remains situated at the point of application. As a mild surfactant, the gel increases antisense ODN penetration and supplies ODNs to the embryo continually for 12-18 h. We have shown a strong decrease in Cx43 protein expression after application of specific antisense oligonucleotides but the abundance of a closely related protein, Connexin-32 (Cx32), was not affected. Application of antisense Cx43 ODNs at stages 8-15 HH before limb outgrowth resulted in dramatic limb phenotypes. About 40% of treated embryos exhibited defects such as truncation of the limb bud, fragmentation into two or more domains, or complete splitting of the limb bud into two or three branches. Molecular analysis of antisense treated embryos failed to detect Shh or Bmp-2 in anterior structures and suggested that extra lobes seen in nicked and split limbs were not a result of establishment of new signalling centres as found after the application of FGF to the flank. However, examination of markers for the AER showed a number of abnormalities. In severely truncated specimens we were unable to detect the expression of either Fgf-4 or Fgf-8. In both nicked and split limbs the expression of these genes was discontinuous. Down-regulation of Cx43 after the antisense application could be comparable to AER removal and results in distal truncation of the limb bud. Taken together these data suggest the existence of a feedback loop between the FGFs and signalling mediated by Cx43.

  11. Exogenous connexin43-expressing autologous skeletal myoblasts ameliorate mechanical function and electrical activity of the rabbit heart after experimental infarction.

    PubMed

    Antanavičiūtė, Ieva; Ereminienė, Eglė; Vysockas, Vaidas; Račkauskas, Mindaugas; Skipskis, Vilius; Rysevaitė, Kristina; Treinys, Rimantas; Benetis, Rimantas; Jurevičius, Jonas; Skeberdis, Vytenis A

    2015-02-01

    Acute myocardial infarction is one of the major causes of mortality worldwide. For regeneration of the rabbit heart after experimentally induced infarction we used autologous skeletal myoblasts (SMs) due to their high proliferative potential, resistance to ischaemia and absence of immunological and ethical concerns. The cells were characterized with muscle-specific and myogenic markers. Cell transplantation was performed by injection of cell suspension (0.5 ml) containing approximately 6 million myoblasts into the infarction zone. The animals were divided into four groups: (i) no injection; (ii) sham injected; (iii) injected with wild-type SMs; and (iv) injected with SMs expressing connexin43 fused with green fluorescent protein (Cx43EGFP). Left ventricular ejection fraction (LVEF) was evaluated by 2D echocardiography in vivo before infarction, when myocardium has stabilized after infarction, and 3 months after infarction. Electrical activity in the healthy and infarction zones of the heart was examined ex vivo in Langendorff-perfused hearts by optical mapping using di-4-ANEPPS, a potential sensitive fluorescent dye. We demonstrate that SMs in the coculture can couple electrically not only to abutted but also to remote acutely isolated allogenic cardiac myocytes through membranous tunnelling tubes. The beneficial effect of cellular therapy on LVEF and electrical activity was observed in the group of animals injected with Cx43EGFP-expressing SMs. L-type Ca(2+) current amplitude was approximately fivefold smaller in the isolated SMs compared to healthy myocytes suggesting that limited recovery of LVEF may be related to inadequate expression or function of L-type Ca(2+) channels in transplanted differentiating SMs. PMID:25529770

  12. Enhanced Transmural Fiber Rotation and Connexin 43 Heterogeneity Are Associated With an Increased Upper Limit of Vulnerability in a Transgenic Rabbit Model of Human Hypertrophic Cardiomyopathy

    PubMed Central

    Ripplinger, Crystal M.; Li, Wenwen; Hadley, Jennifer; Chen, Junjie; Rothenberg, Florence; Lombardi, Raffaella; Wickline, Samuel A.; Marian, Ali J.; Efimov, Igor R.

    2008-01-01

    Human hypertrophic cardiomyopathy, characterized by cardiac hypertrophy and myocyte disarray, is the most common cause of sudden cardiac death in the young. Hypertrophic cardiomyopathy is often caused by mutations in sarcomeric genes. We sought to determine arrhythmia propensity and underlying mechanisms contributing to arrhythmia in a transgenic (TG) rabbit model (β-myosin heavy chain–Q403) of human hypertrophic cardiomyopathy. Langendorff-perfused hearts from TG (n = 6) and wild-type (WT) rabbits (n = 6) were optically mapped. The upper and lower limits of vulnerability, action potential duration (APD) restitution, and conduction velocity were measured. The transmural fiber angle shift was determined using diffusion tensor MRI. The transmural distribution of connexin 43 was quantified with immunohistochemistry. The upper limit of vulnerability was significantly increased in TG versus WT hearts (13.3 ± 2.1 versus 7.4 ± 2.3 V/cm; P = 3.2e−5), whereas the lower limits of vulnerability were similar. APD restitution, conduction velocities, and anisotropy were also similar. Left ventricular transmural fiber rotation was significantly higher in TG versus WT hearts (95.6 ± 10.9° versus 79.2 ± 7.8°; P = 0.039). The connexin 43 density was significantly increased in the mid-myocardium of TG hearts compared with WT (5.46 ± 2.44% versus 2.68 ± 0.77%; P = 0.024), and similar densities were observed in the endo- and epicardium. Because a nearly 2-fold increase in upper limit of vulnerability was observed in the TG hearts without significant changes in APD restitution, conduction velocity, or the anisotropy ratio, we conclude that structural remodeling may underlie the elevated upper limit of vulnerability in human hypertrophic cardiomyopathy. PMID:17885214

  13. Investigation of connexin 43 uncoupling and prolongation of the cardiac QRS complex in preclinical and marketed drugs

    PubMed Central

    Burnham, M P; Sharpe, P M; Garner, C; Hughes, R; Pollard, C E; Bowes, J

    2014-01-01

    Background and Purpose Prolongation of the cardiac QRS complex is linked to increased mortality and may result from drug-induced inhibition of cardiac sodium channels (hNaV1.5). There has been no systematic evaluation of preclinical and marketed drugs for their additional potential to cause QRS prolongation via gap junction uncoupling. Experimental Approach Using the human cardiac gap junction connexin 43 (hCx43), a dye transfer ‘parachute’ assay to determine IC50 values for compound ranking was validated with compounds known to uncouple gap junctions. Uncoupling activity (and hNaV1.5 inhibition by automated patch clamp) was determined in a set of marketed drugs and preclinical candidate drugs, each with information regarding propensity to prolong QRS. Key Results The potency of known gap junction uncouplers to uncouple hCx43 was ranked (according to IC50) as phorbol ester>digoxin>meclofenamic acid>carbenoxolone>heptanol. Among the drugs associated with QRS prolongation, 29% were found to uncouple hCx43 (IC50 < 50 μM), whereas no uncoupling activity was observed in drugs not associated with QRS prolongation. In preclinical candidate drugs, hCx43 and hNaV1.5 IC50 values were similar (within threefold). No consistent margin over preclinical Cmax (free) was apparent for QRS prolongation associated with Cx43 inhibition. However, instances were found of QRS prolonging compounds that uncoupled hCx43 with significantly less activity at hNaV1.5. Conclusion and Implications These results demonstrate that off-target uncoupling activity is apparent in drug and drug-like molecules. Although the full ramifications of Cx inhibition remain to be established, screening for hCx43 off-target activity could reduce the likelihood of developing candidate drugs with a risk of causing QRS prolongation. PMID:24328991

  14. Effects of space flight on the immunohistochemical demonstration of connexin 26 and connexin 43 in the postpartum uterus of rats

    NASA Technical Reports Server (NTRS)

    Burden, H. W.; Zary, J.; Alberts, J. R.

    1999-01-01

    The effect of space flight in a National Aeronautics and Space Administration shuttle was studied in pregnant rats. Rats were launched on day 11 of gestation and recovered on day 20 of gestation. Pregnancy was allowed to proceed to term and rats delivered vaginally on days 22-23, although flight animals required more labour contractions to complete the delivery process. Pups were placed with foster dams and connexin 26 and 43 were examined in the uterus of flight animals approximately 3 h after delivery. Space flight did not affect uterine connexin 26, localized primarily in epithelial cells of the endometrium, but decreased connexin 43, the major gap junction protein in the myometrium. It is suggested that decreased connexin 43 alters synchronization and coordination of labour contractions, resulting in a requirement for more contractions to complete the delivery process.

  15. Characteristics of the Localization of Connexin 43 in Satellite Cells during Skeletal Muscle Regeneration In Vivo.

    PubMed

    Ishido, Minenori; Kasuga, Norikatsu

    2015-04-28

    For myogenesis, new myotubes are formed by the fusion of differentiated myoblasts. In the sequence of events for myotube formation, intercellular communication through gap junctions composed of connexin 43 (Cx43) plays critical roles in regulating the alignment and fusion of myoblasts in advances of myotube formation in vitro. On the other hand, the relationship between the expression patterns of Cx43 and the process of myotube formation in satellite cells during muscle regeneration in vivo remains poorly understood. The present study investigated the relationship between Cx43 and satellite cells in muscle regeneration in vivo. The expression of Cx43 was detected in skeletal muscles on day 1 post-muscle injury, but not in control muscles. Interestingly, the expression of Cx43 was not localized on the inside of the basement membrane of myofibers in the regenerating muscles. Moreover, although the clusters of differentiated satellite cells, which represent a more advanced stage of myotube formation, were observed on the inside of the basement membrane of myofibers in regenerating muscles, the expression of Cx43 was not localized in the clusters of these satellite cells. Therefore, in the present study, it was suggested that Cx43 may not directly contribute to muscle regeneration via satellite cells. PMID:26019374

  16. Prostaglandin E2 and Connexin 43 crosstalk in the osteogenesis induced by extracorporeal shockwave.

    PubMed

    Chen, Youbin; Xu, Jiankun; Liao, Haojie; Ma, Zebin; Zhang, Yuantao; Chen, Hongjiang; Huang, Zhonglian; Hu, Jun

    2016-09-01

    As a type of mechanical stimulation, extracorporeal shockwave (ESW) has been widely used in the clinic to treat bone fracture delayed union and non-unions. A large number of studies have shown beneficial effects of ESW in promoting fracture healing by inducing bone regeneration; however, the underlying mechanisms remain unclear. ESW has been shown to induce the production of prostaglandin E2 (PGE2), which is essential for gap junction intercellular communication in response to mechanical stress. Among the 19 known gap junction subunits, connexin43 (Cx43) is the most prevalent for mediating the response of mechanical stress. However, to our knowledge, the effect of ESW on Cx43 expression has not been reported before. Herein, we propose that a crosstalk between PGE2 and Cx43 is involved in the enhancement of osteogenesis induced by ESW. We review the currently available data to propose an unrevealed, but important mechanism via which ESW treatment affects osteogenic differentiation of bone marrow stromal cells.

  17. Molecular cloning and evolutionary analysis of the GJA1 (connexin43) gene from bats (Chiroptera).

    PubMed

    Wang, Li; Li, Gang; Wang, Jinhong; Ye, Shaohui; Jones, Gareth; Zhang, Shuyi

    2009-04-01

    Gap junction protein connexin43 (Cx43), encoded by the GJA1 gene, is the most abundant connexin in the cardiovascular system and was reported as a crucial factor maintaining cardiac electrical conduction, as well as having a very important function in facilitating the recycling of potassium ions from hair cells in the cochlea back into the cochlear endolymph during auditory transduction processes. In mammals, bats are the only taxon possessing powered flight, placing exceptional demand on many organismal processes. To meet the demands of flying, the hearts of bats show many specialties. Moreover, ultrasonic echolocation allows bat species to orientate and often detect and locate food in darkness. In this study, we cloned the full-length coding region of GJA1 gene from 12 different species of bats and obtained orthologous sequences from other mammals. We used the maximum likelihood method to analyse the evolution of GJA1 gene in mammals and the lineage of bats. Our results showed this gene is much conserved in mammals, as well as in bats' lineage. Compared with other mammals, we found one private amino acid substitution shared by bats, which is located on the inner loop domain, as well as some species-specific amino acid substitutions. The evolution rate analyses showed the signature of purifying selection on not only different classification level lineages but also the different domains and amino acid residue sites of this gene. Also, we suggested that GJA1 gene could be used as a good molecular marker to do the phylogenetic reconstruction.

  18. Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function.

    PubMed

    Sharma, Parveen; Abbasi, Cynthia; Lazic, Savo; Teng, Allen C T; Wang, Dingyan; Dubois, Nicole; Ignatchenko, Vladimir; Wong, Victoria; Liu, Jun; Araki, Toshiyuki; Tiburcy, Malte; Ackerley, Cameron; Zimmermann, Wolfram H; Hamilton, Robert; Sun, Yu; Liu, Peter P; Keller, Gordon; Stagljar, Igor; Scott, Ian C; Kislinger, Thomas; Gramolini, Anthony O

    2015-01-01

    Membrane proteins are crucial to heart function and development. Here we combine cationic silica-bead coating with shotgun proteomics to enrich for and identify plasma membrane-associated proteins from primary mouse neonatal and human fetal ventricular cardiomyocytes. We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts. Functional analysis of Tmem65 both in vitro using lentiviral shRNA-mediated knockdown in mouse cardiomyocytes and in vivo using morpholino-based knockdown in zebrafish show marked alterations in gap junction function and cardiac morphology. Molecular analyses suggest that Tmem65 interaction with connexin 43 (Cx43) is required for correct localization of Cx43 to the intercalated disc, since Tmem65 deletion results in marked internalization of Cx43, a shorter half-life through increased degradation, and loss of Cx43 function. Our data demonstrate that the membrane protein Tmem65 is an intercalated disc protein that interacts with and functionally regulates ventricular Cx43.

  19. Effect of enhanced expression of connexin 43 on sunitinib-induced cytotoxicity in mesothelioma cells.

    PubMed

    Uzu, Miaki; Sato, Hiromi; Yamada, Ryota; Kashiba, Tatsuro; Shibata, Yukihiro; Yamaura, Katsunori; Ueno, Koichi

    2015-05-01

    Connexin (Cx) makes up a type of intercellular channel called gap junction (GJ). GJ plays a regulatory role in cellular physiology. The Cx expression level is often decreased in cancer cells compared to that in healthy ones, and the restoration of its expression has been shown to exert antiproliferative effects. This work aims to evaluate the effect of the restoration of connexin 43 (Cx43) (the most ubiquitous Cx subtype) expression on sunitinib (SU)-induced cytotoxicity in malignant mesothelioma (MM) cells. Increased Cx43 expression in an MM cell line (H28) improved the ability of SU to inhibit receptor tyrosine kinase (RTK) signaling. Moreover, higher Cx43 expression promoted SU-induced apoptosis. The cell viability test revealed that Cx43 enhanced the cytotoxic effect of SU in a GJ-independent manner. The effect of Cx43 on a proapoptotic factor, Bax, was then investigated. The interaction between Cx43 and Bax was confirmed by immunoprecipitation. Furthermore, higher Cx43 expression increased the production of a cleaved (active) form of Bax during SU-induced apoptosis with no alteration in total Bax expression. These findings indicate that Cx43 most likely increases sensitivity to SU in H28 through direct interaction with Bax. In conclusion, we found that Cx43 overcame the chemoresistance of MM cells.

  20. Phosphorylation at Connexin43 Serine-368 Is Necessary for Myocardial Conduction During Metabolic Stress.

    PubMed

    Nassal, Michelle M J; Werdich, Andreas A; Wan, Xiaoping; Hoshi, Malcolm; Deschênes, Isabelle; Rosenbaum, David S; Donahue, J Kevin

    2016-01-01

    Connexin43 (Cx43) phosphorylation alters gap junction localization and function. In particular, phosphorylation at serine-368 (S368) has been suggested to alter gap junctional conductance, but previous reports have shown inconsistent results for both timing and functional effects of S368 phosphorylation. The objective of this study was to determine the functional effects of isolated S368 phosphorylation. We evaluated wild-type Cx43 (AdCx43) and mutations simulating permanent phosphorylation (Ad368E) or preventing phosphorylation (Ad368A) at S368. Function was assessed by optical mapping of electrical conduction in patterned cultures of neonatal rat ventricular myocytes, under baseline and metabolic stress (MS) conditions. Baseline conduction velocity (CV) was similar for all groups. In the AdCx43 and Ad368E groups, MS moderately decreased CV. Ad368A caused complete conduction block during MS. Triton-X solubility assessment showed no change in Cx43 location during conduction impairment. Western blot analysis showed that Cx43-S368 phosphorylation was present at baseline, and that it decreased during MS. Our data indicate that phosphorylation at S368 does not affect CV under baseline conditions, and that preventing S368 phosphorylation makes Cx43 hypersensitive to MS. These results show the critical role of S368 phosphorylation during stress conditions.

  1. Common mechanisms linking connexin43 to neural progenitor cell migration and glioma invasion.

    PubMed

    Naus, Christian C; Aftab, Qurratulain; Sin, Wun Chey

    2016-02-01

    Cell migration is critical for cell differentiation, tissue formation and organ development. Several mechanisms come to play in the process of cell migration, orchestrating changes in cell polarity, adhesion, process extension and motility. Recent findings have shown that gap junctions, and specifically connexin43 (Cx43), can play a significant role in these processes, impacting adhesion and cytoskeletal rearrangements. Thus Cx43 within a cell regulates its motility and migration via intracellular signaling. Furthermore, Cx43 in the host cells can impact the degree of cellular migration through that tissue. Similarities in these connexin-based processes account for both neural progenitor migration in the developing brain, and for glioma cell invasion in the mature brain. In both cases, Cx43 in the tissue ("soil") in which cells ("seeds") exist facilitates their migration and, for glioma cells, tissue invasion. Cx43 mediates these effects through channel- and non-channel-dependent mechanisms which have similarities in both paradigms of cell migration. This provides insight into developmental processes and pathological situations, as well as possible therapeutic approaches regarding specific functional domains of gap junction proteins.

  2. Involvement of urinary bladder Connexin43 and the circadian clock in coordination of diurnal micturition rhythm

    PubMed Central

    Negoro, Hiromitsu; Kanematsu, Akihiro; Doi, Masao; Suadicani, Sylvia O.; Matsuo, Masahiro; Imamura, Masaaki; Okinami, Takeshi; Nishikawa, Nobuyuki; Oura, Tomonori; Matsui, Shigeyuki; Seo, Kazuyuki; Tainaka, Motomi; Urabe, Shoichi; Kiyokage, Emi; Todo, Takeshi; Okamura, Hitoshi; Tabata, Yasuhiko; Ogawa, Osamu

    2012-01-01

    Summary Nocturnal enuresis in children and nocturia in the elderly are two highly prevalent clinical conditions characterized by a mismatch between urine production rate in the kidneys and storage in the urinary bladder during the sleep phase. Here we demonstrate, using a novel method for automated recording of mouse micturition, that connexin43 (Cx43), a bladder gap junction protein, is a negative regulator of functional bladder capacity. Bladder Cx43 levels and functional capacity show circadian oscillations in wild-type mice, but such rhythms are completely lost in Cry-null mice having a dysfunctional biological clock. Bladder muscle cells have an internal clock, and show oscillations of Cx43 and gap junction function. A clock regulator, Rev-erbα, upregulates Cx43 transcription as a co-factor of Sp1 using Sp1 cis-elements of the promoter. Therefore, circadianoscillation of Cx43 is associated with the biological clock and contributes to diurnal changes in bladder capacity, which avoids disturbance of sleep by micturition. PMID:22549838

  3. Connexin 43 expression is associated with poor survival in patients with esophageal squamous cell carcinoma

    PubMed Central

    TANAKA, TATSUYA; KIMURA, MASAHIRO; ISHIGURO, HIDEYUKI; MIZOGUCHI, KOUJI; TAKEYAMA, HIROMITSU

    2016-01-01

    Connexin 43 (Cx43) is an important gap junction protein in vertebrates, which has been reported to function as a tumor suppressor in a number of organs. However, the mechanism underlying the effect of Cx43 on tumor progression remains unknown, with only a limited number of studies reporting on the role of Cx43 in esophageal cancer. In the present study, Cx43 expression was analyzed by immunohistochemical staining and the associations between Cx43 expression and clinicopathological characteristics or prognosis were evaluated. Cx43 was expressed at a high frequency in patients with esophageal squamous cell carcinoma (ESCC). Of the 98 ESCC cases investigated, positivity for Cx43 was observed in 62 cases (63.26%). In patients with high Cx43 expression, the survival rates were significantly reduced compared with those in patients with low Cx43 expression. Moreover, the overexpression of Cx43, as measured by immunohistochemistry, was an independent prognostic indicator of ESCC. Thus, our data indicated that Cx43 may be a candidate molecular prognostic marker and molecular target for the development of an effective therapeutic intervention for patients with esophageal cancer. PMID:27284434

  4. The cardiac connexome: Non-canonical functions of connexin43 and their role in cardiac arrhythmias.

    PubMed

    Leo-Macias, Alejandra; Agullo-Pascual, Esperanza; Delmar, Mario

    2016-02-01

    Connexin43 is the major component of gap junctions, an anatomical structure present in the cardiac intercalated disc that provides a low-resistance pathway for direct cell-to-cell passage of electrical charge. Recent studies have shown that in addition to its well-established function as an integral membrane protein that oligomerizes to form gap junctions, Cx43 plays other roles that are independent of channel (or perhaps even hemi-channel) formation. This article discusses non-canonical functions of Cx43. In particular, we focus on the role of Cx43 as a part of a protein interacting network, a connexome, where molecules classically defined as belonging to the mechanical junctions, the gap junctions and the sodium channel complex, multitask and work together to bring about excitability, electrical and mechanical coupling between cardiac cells. Overall, viewing Cx43 as a multi-functional protein, beyond gap junctions, opens a window to better understand the function of the intercalated disc and the pathological consequences that may result from changes in the abundance or localization of Cx43 in the intercalated disc subdomain. PMID:26673388

  5. Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries

    USGS Publications Warehouse

    Melton, C.M.; Zaunbrecher, G.M.; Yoshizaki, G.; Patio, R.; Whisnant, S.; Rendon, A.; Lee, V.H.

    2001-01-01

    A major form of cell-cell communication is mediated by gap junctions, aggregations of intercellular channels composed of connexins (Cxs), which are responsible for exchange of low molecular weight (< 1200 Da) cytosolic materials. These channels are a growing family of related proteins. This study was designed to determine the ontogeny of connexin 43 (Cx43) during early stages of follicular development in prepubertal porcine ovaries. A partial-length (412 base) cDNA clone was obtained from mature porcine ovaries and determined to have 98% identity with published porcine Cx43. Northern blot analysis demonstrated a 4.3-kb mRNA in total RNA isolated from prepubertal and adult porcine ovaries. In-situ hybridization revealed that Cx43 mRNA was detectable in granulosa cells of primary follicles but undetectable in dormant primordial follicles. The intensity of the signal increased with follicular growth and was greatest in the large antral follicles. Immunohistochemical evaluation indicated that Cx43 protein expression correlated with the presence of Cx43 mRNA. These results indicate that substantial amounts of Cx43 are first expressed in granulosa cells following activation of follicular development and that this expression increases throughout follicular growth and maturation. These findings suggest an association between the enhancement of intercellular gap-junctional communication and onset of follicular growth. ?? 2001 Elsevier Science Inc. All rights reserved.

  6. The modulatory effects of connexin 43 on cell death/survival beyond cell coupling.

    PubMed

    Rodríguez-Sinovas, Antonio; Cabestrero, Alberto; López, Diego; Torre, Iratxe; Morente, Miriam; Abellán, Arancha; Miró, Elisabet; Ruiz-Meana, Marisol; García-Dorado, David

    2007-01-01

    Connexins form a diverse and ubiquitous family of integral membrane proteins. Characteristically, connexins are assembled into intercellular channels that aggregate into discrete cell-cell contact areas termed gap junctions (GJ), allowing intercellular chemical communication, and are essential for propagation of electrical impulses in excitable tissues, including, prominently, myocardium, where connexin 43 (Cx43) is the most important isoform. Previous studies have shown that GJ-mediated communication has an important role in the cellular response to stress or ischemia. However, recent evidence suggests that connexins, and in particular Cx43, may have additional effects that may be important in cell death and survival by mechanisms independent of cell to cell communication. Connexin hemichannels, located at the plasma membrane, may be important in paracrine signaling that could influence intracellular calcium and cell survival by releasing intracellular mediators as ATP, NAD(+), or glutamate. In addition, recent studies have shown the presence of connexins in cell structures other than the plasma membrane, including the cell nucleus, where it has been suggested that Cx43 influences cell growth and differentiation. In addition, translocation of Cx43 to mitochondria appears to be important for certain forms of cardioprotection. These findings open a new field of research of previously unsuspected roles of Cx43 intracellular signaling.

  7. Cloning, sequence analysis and phylogeny of connexin43 isolated from American black bear heart.

    PubMed

    Van Der Heyden, Marcel A G; Kok, Bart; Kouwenhoven, Evelyn N; Toien, Oivind; Barnes, Brian M; Fedorov, Vadim G; Efimov, Igor R; Opthof, Tobias

    2007-10-01

    Conduction in the heart requires gap junctions. In mammalian ventricular myocytes these consist of connexin43 (Cx43). Hearts of non-hibernating species display conduction disturbances at reduced temperatures. These may exacerbate into lethal arrhythmias. Hibernating species are protected against these arrhythmias by a non-resolved mechanism. To analyze whether the amino acid composition of Cx43 from the hibernating American black bear displays specific features, we cloned the full coding sequence of Ursus americanus Cx43 and compared with that of other (non)hibernating species. UaCx43 displays 99.7% identity to rabbit Cx43 at the amino acid level. No specific features were observed in UaCx43 when compared to previously cloned Cx43 from hibernating and non-hibernating mammals. Phylogenetic tree reconstruction of this and other published full-length Cx43 sequences reveals a very high level of conservation from fish to men. Finally, one of the previously identified six mammalian characteristic amino acids, is not conserved in the black bear. PMID:17654014

  8. Prostaglandin E2 and Connexin 43 crosstalk in the osteogenesis induced by extracorporeal shockwave.

    PubMed

    Chen, Youbin; Xu, Jiankun; Liao, Haojie; Ma, Zebin; Zhang, Yuantao; Chen, Hongjiang; Huang, Zhonglian; Hu, Jun

    2016-09-01

    As a type of mechanical stimulation, extracorporeal shockwave (ESW) has been widely used in the clinic to treat bone fracture delayed union and non-unions. A large number of studies have shown beneficial effects of ESW in promoting fracture healing by inducing bone regeneration; however, the underlying mechanisms remain unclear. ESW has been shown to induce the production of prostaglandin E2 (PGE2), which is essential for gap junction intercellular communication in response to mechanical stress. Among the 19 known gap junction subunits, connexin43 (Cx43) is the most prevalent for mediating the response of mechanical stress. However, to our knowledge, the effect of ESW on Cx43 expression has not been reported before. Herein, we propose that a crosstalk between PGE2 and Cx43 is involved in the enhancement of osteogenesis induced by ESW. We review the currently available data to propose an unrevealed, but important mechanism via which ESW treatment affects osteogenic differentiation of bone marrow stromal cells. PMID:27515217

  9. Modulatory Effects of Connexin-43 Expression on Gap Junction Intercellular Communications with Mast Cells and Fibroblasts

    PubMed Central

    Pistorio, Ashey L.; Ehrlich, H. Paul

    2011-01-01

    The influence of mast cells upon aberrant wound repair and excessive fibrosis has supportive evidence, but the mechanism for these mast cell activities is unclear. It is proposed that heterocellular gap junctional intercellular communication (GJIC) between fibroblasts and mast cells directs some fibroblast activities. An in vitro model was used employing a rodent derived peritoneal mast cell line (RMC-1) and human dermal derived fibroblasts. The influence of the expression of the gap junction channel structural protein, connexin 43 (Cx-43) on heterocellular GJIC, the expression of microtubule β-tubulin and microfilament α smooth muscle actin (SMA) were investigated. The knockdown of Cx-43 by siRNA in RMC-1 cells completely blocked GJIC between RMC-1 cells. SiRNA knockdown of Cx-43 within fibroblasts only dampened GJIC between fibroblasts. It appears Cx-43 is the only expressed connexin in RMC-1 cells. Fibroblasts express other connexins that participate in GJIC between fibroblasts in the absence of Cx-43 expression. Heterocellular GJIC between RMC-1 cells and fibroblasts transformed fibroblasts into myofibroblasts, expressing α SMA within cytoplasmic stress fibers. The knockdown of Cx-43 in RMC-1 cells increased β-tubulin expression, but its knockdown in fibroblasts reduced β-tubulin expression. Knocking down the expression of Cx-43 in fibroblasts limited α SMA expression. Cx-43 participation is critical for heterocellular GJIC between mast cells and fibroblasts, which may herald a novel direction for controlling fibrosis. PMID:21328609

  10. Connexin 43 Mediates White Adipose Tissue Beiging by Facilitating the Propagation of Sympathetic Neuronal Signals.

    PubMed

    Zhu, Yi; Gao, Yong; Tao, Caroline; Shao, Mengle; Zhao, Shangang; Huang, Wei; Yao, Ting; Johnson, Joshua A; Liu, Tiemin; Cypess, Aaron M; Gupta, Olga; Holland, William L; Gupta, Rana K; Spray, David C; Tanowitz, Herbert B; Cao, Lei; Lynes, Matthew D; Tseng, Yu-Hua; Elmquist, Joel K; Williams, Kevin W; Lin, Hua V; Scherer, Philipp E

    2016-09-13

    "Beige" adipocytes reside in white adipose tissue (WAT) and dissipate energy as heat. Several studies have shown that cold temperature can activate pro-opiomelanocortin-expressing (POMC) neurons and increase sympathetic neuronal tone to regulate WAT beiging. WAT, however, is traditionally known to be sparsely innervated. Details regarding the neuronal innervation and, more importantly, the propagation of the signal within the population of "beige" adipocytes are sparse. Here, we demonstrate that beige adipocytes display an increased cell-to-cell coupling via connexin 43 (Cx43) gap junction channels. Blocking of Cx43 channels by 18α-glycyrrhetinic acid decreases POMC-activation-induced adipose tissue beiging. Adipocyte-specific deletion of Cx43 reduces WAT beiging to a level similar to that observed in denervated fat pads. In contrast, overexpression of Cx43 is sufficient to promote beiging even with mild cold stimuli. These data reveal the importance of cell-to-cell communication, effective in cold-induced WAT beiging, for the propagation of limited neuronal inputs in adipose tissue. PMID:27626200

  11. Two Different Functions of Connexin43 Confer Two Different Bone Phenotypes in Zebrafish.

    PubMed

    Misu, Akihiro; Yamanaka, Hiroaki; Aramaki, Toshihiro; Kondo, Shigeru; Skerrett, I Martha; Iovine, M Kathryn; Watanabe, Masakatsu

    2016-06-10

    Fish remain nearly the same shape as they grow, but there are two different modes of bone growth. Bones in the tail fin (fin ray segments) are added distally at the tips of the fins and do not elongate once produced. On the other hand, vertebrae enlarge in proportion to body growth. To elucidate how bone growth is controlled, we investigated a zebrafish mutant, steopsel (stp(tl28d)). Vertebrae of stp(tl28d) (/+) fish look normal in larvae (∼30 days) but are distinctly shorter (59-81%) than vertebrae of wild type fish in adults. In contrast, the lengths of fin rays are only slightly shorter (∼95%) than those of the wild type in both larvae and adults. Positional cloning revealed that stp encodes Connexin43 (Cx43), a connexin that functions as a gap junction and hemichannel. Interestingly, cx43 was also identified as the gene causing the short-of-fin (sof) phenotype, in which the fin ray segments are shorter but the vertebrae are normal. To identify the cause of this difference between the alleles, we expressed Cx43 exogenously in Xenopus oocytes and performed electrophysiological analysis of the mutant proteins. Gap junction coupling induced by Cx43(stp) or Cx43(sof) was reduced compared with Cx43-WT. On the other hand, only Cx43(stp) induced abnormally high (50× wild type) transmembrane currents through hemichannels. Our results suggest that Cx43 plays critical and diverse roles in zebrafish bone growth.

  12. Targeting connexin 43 protects against the progression of experimental chronic kidney disease in mice.

    PubMed

    Abed, Ahmed; Toubas, Julie; Kavvadas, Panagiotis; Authier, Florence; Cathelin, Dominique; Alfieri, Carlo; Boffa, Jean-Jacques; Dussaule, Jean-Claude; Chatziantoniou, Christos; Chadjichristos, Christos E

    2014-10-01

    Excessive recruitment of monocytes and progression of fibrosis are hallmarks of chronic kidney disease (CKD). Recently we reported that the expression of connexin 43 (Cx43) was upregulated in the kidney during experimental nephropathy. To investigate the role of Cx43 in the progression of CKD, we interbred RenTg mice, a genetic model of hypertension-induced CKD, with Cx43+/- mice. The renal cortex of 5-month-old RenTgCx43+/- mice showed a marked decrease of cell adhesion markers leading to reduced monocyte infiltration and interstitial renal fibrosis compared with their littermates. In addition, functional and histological parameters such as albuminuria and glomerulosclerosis were ameliorated in RenTgCx43+/- mice. Interestingly, treatment with Cx43 antisense produced remarkable improvement of renal function and structure in 1-year-old RenTg mice. Similar results were found in Cx43+/- or wild-type mice treated with Cx43 antisense after obstructive nephropathy. Furthermore, in these mice, Cx43 antisense attenuated E-cadherin downregulation and phosphorylation of the transcription factor Sp1 by the ERK pathway resulting in decreased transcription of type I collagen gene. Interestingly, Cx43-specific blocking peptide inhibited monocyte adhesion in activated endothelium and profibrotic pathways in tubular cells. Cx43 was highly increased in biopsies of patients with CKD. Thus, Cx43 may represent a new therapeutic target against the progression of CKD.

  13. Connexin 43 controls the multipolar phase of neuronal migration to the cerebral cortex.

    PubMed

    Liu, Xiuxin; Sun, Lin; Torii, Masaaki; Rakic, Pasko

    2012-05-22

    The prospective pyramidal neurons, migrating from the proliferative ventricular zone to the overlaying cortical plate, assume multipolar morphology while passing through the transient subventricular zone. Here, we show that this morphogenetic transformation, from the bipolar to the mutipolar and then back to bipolar again, is associated with expression of connexin 43 (Cx43) and, that knockdown of Cx43 retards, whereas its overexpression enhances, this morphogenetic process. In addition, we have observed that knockdown of Cx43 reduces expression of p27, whereas overexpression of p27 rescues the effect of Cx43 knockdown in the multipolar neurons. Furthermore, functional gap junction/hemichannel domain, and the C-terminal domain of Cx43, independently enhance the expression of p27 and promote the morphological transformation and migration of the multipolar neurons in the SVZ/IZ. Collectively, these results indicate that Cx43 regulates the passage of migrating neurons through their multipolar stage via p27 signaling and that interference with this process, by either genetic and/or environmental factors, may cause cortical malformations. PMID:22566616

  14. Connexin 43 controls the multipolar phase of neuronal migration to the cerebral cortex.

    PubMed

    Liu, Xiuxin; Sun, Lin; Torii, Masaaki; Rakic, Pasko

    2012-05-22

    The prospective pyramidal neurons, migrating from the proliferative ventricular zone to the overlaying cortical plate, assume multipolar morphology while passing through the transient subventricular zone. Here, we show that this morphogenetic transformation, from the bipolar to the mutipolar and then back to bipolar again, is associated with expression of connexin 43 (Cx43) and, that knockdown of Cx43 retards, whereas its overexpression enhances, this morphogenetic process. In addition, we have observed that knockdown of Cx43 reduces expression of p27, whereas overexpression of p27 rescues the effect of Cx43 knockdown in the multipolar neurons. Furthermore, functional gap junction/hemichannel domain, and the C-terminal domain of Cx43, independently enhance the expression of p27 and promote the morphological transformation and migration of the multipolar neurons in the SVZ/IZ. Collectively, these results indicate that Cx43 regulates the passage of migrating neurons through their multipolar stage via p27 signaling and that interference with this process, by either genetic and/or environmental factors, may cause cortical malformations.

  15. Characteristics of the Localization of Connexin 43 in Satellite Cells during Skeletal Muscle Regeneration In Vivo.

    PubMed

    Ishido, Minenori; Kasuga, Norikatsu

    2015-04-28

    For myogenesis, new myotubes are formed by the fusion of differentiated myoblasts. In the sequence of events for myotube formation, intercellular communication through gap junctions composed of connexin 43 (Cx43) plays critical roles in regulating the alignment and fusion of myoblasts in advances of myotube formation in vitro. On the other hand, the relationship between the expression patterns of Cx43 and the process of myotube formation in satellite cells during muscle regeneration in vivo remains poorly understood. The present study investigated the relationship between Cx43 and satellite cells in muscle regeneration in vivo. The expression of Cx43 was detected in skeletal muscles on day 1 post-muscle injury, but not in control muscles. Interestingly, the expression of Cx43 was not localized on the inside of the basement membrane of myofibers in the regenerating muscles. Moreover, although the clusters of differentiated satellite cells, which represent a more advanced stage of myotube formation, were observed on the inside of the basement membrane of myofibers in regenerating muscles, the expression of Cx43 was not localized in the clusters of these satellite cells. Therefore, in the present study, it was suggested that Cx43 may not directly contribute to muscle regeneration via satellite cells.

  16. Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression

    PubMed Central

    Henry, Matthew K.; Welliver, Kathryn C.; Jepson, Amanda J.; Garnett, Emily R.

    2013-01-01

    Hindlimb unloading (HU) is a well-established animal model of cardiovascular deconditioning. Previous data indicate that HU results in cardiac sympathovagal imbalance. It is well established that cardiac sympathovagal imbalance increases the risk for developing cardiac arrhythmias. The cardiac gap junction protein connexin 43 (Cx43) is predominately expressed in the left ventricle (LV) and ensures efficient cell-to-cell electrical coupling. In the current study we wanted to test the hypothesis that HU would result in increased predisposition to cardiac arrhythmias and alter the expression and/or phosphorylation of LV-Cx43. Electrocardiographic data using implantable telemetry were obtained over a 10- to 14-day HU or casted control (CC) condition and in response to a sympathetic stressor using isoproterenol administration and brief restraint. The arrhythmic burden was calculated using a modified scoring system to quantify spontaneous and provoked arrhythmias. In addition, Western blot analysis was used to measure LV-Cx43 expression in lysates probed with antibodies directed against the total and an unphosphorylated form of Cx43 in CC and HU rats. HU resulted in a significantly greater total arrhythmic burden during the sympathetic stressor with significantly more ventricular arrhythmias occurring. In addition, there was increased expression of total LV-Cx43 observed with no difference in the expression of unphosphorylated LV-Cx43. Specifically, the increased expression of LV-Cx43 was consistent with the phosphorylated form. These data taken together indicate that cardiovascular deconditioning produced through HU results in increased predisposition to cardiac arrhythmias and increased expression of phosphorylated LV-Cx43. PMID:23302960

  17. Association of reduced Connexin 43 expression with retinal vascular lesions in human diabetic retinopathy.

    PubMed

    Tien, Thomas; Muto, Tetsuya; Zhang, Joyce; Sohn, Elliott H; Mullins, Robert F; Roy, Sayon

    2016-05-01

    Connexin 43 (Cx43) downregulation promotes apoptosis in retinal vascular cells of diabetic animal models; however, its relevance to human diabetic retinopathy has not been established. In this study, we investigated whether diabetes alters Cx43 expression and promotes retinal vascular lesions in human retinas. Diabetic human eyes (aged 64-94 years) and non-diabetic human eyes (aged 61-90 years) were analyzed in this study. Retinal protein samples and retinal capillary networks were assessed for Cx43 level by Western blot (WB) analysis and immunostaining. In parallel, retinal capillary networks were stained with hematoxylin and periodic acid Schiff to determine the extent of pericyte loss (PL) and acellular capillaries (AC) in these retinas. Cx43 protein expression was significantly reduced in the diabetic retinas compared to non-diabetic retinas as indicated by WB analysis (81 ± 11% of control). Additionally, a significant decrease in the number of Cx43 plaques per unit length of vessel was observed in the diabetic retinas compared to those of non-diabetic retinas (62 ± 10% of control; p < 0.005). Importantly, a strong inverse relationship was noted between Cx43 expression and the relative number of AC (r = -0.89; p < 0.0005), and between Cx43 expression and number of pericyte loss (r = -0.88; p < 0.0005). Overall, these results show that Cx43 expression is reduced in the human diabetic retinas and Cx43 reduction is associated with increased vascular cell death. These findings suggest that diabetes decreases retinal Cx43 expression and that the development of PL and AC is associated with reduced Cx43 expression in human diabetic retinopathy. PMID:26738943

  18. The role of connexin43-Src interaction in astrocytomas: A molecular puzzle.

    PubMed

    Tabernero, A; Gangoso, E; Jaraíz-Rodríguez, M; Medina, J M

    2016-05-26

    Connexin43 (Cx43) as a building block of gap junction channels and hemichannels exerts important functions in astrocytes. When these cells acquire a malignant phenotype Cx43 protein but not mRNA levels are downregulated, being negligible in high-grade astrocytoma or glioblastoma multiforme, the most common and deadliest of malignant primary brain tumors in adults. Some microRNAs associated to glioma target Cx43 and could explain the lack of correlation between mRNA and protein levels of Cx43 found in some high-grade astrocytomas. More importantly, these microRNAs could be a promising therapeutic target. A great number of studies have confirmed the relationship between cancer and connexins that was proposed by Loewenstein more than 40years ago, but these studies have also revealed that this is a very complex relationship. Indeed, restoring Cx43 to glioma cells reduces their rate of proliferation and their tumorigenicity but this tumor suppressor effect could be counterbalanced by its effects on invasiveness, adhesion and migration. The mechanisms underlying these effects suggest the participation of a great variety of proteins that bind to different regions of Cx43. The present review focuses on an intrinsically disordered region of the C-terminal domain of Cx43 in which converges the interaction of several proteins, including the proto-oncogene Src. We summarize data that indicate that Cx43-Src interaction inhibits the oncogenic activity of Src and promotes a conformational change in the structure of Cx43 that allosterically modifies the binding to other important signaling proteins. As a consequence, crucial cell functions, such as proliferation or migration, could be strongly affected. We propose that the knowledge of the structural basis of the antitumorigenic effect of Cx43 on astrocytomas could help to design new therapies against this incurable disease. PMID:25711938

  19. The role of connexin43-Src interaction in astrocytomas: A molecular puzzle.

    PubMed

    Tabernero, A; Gangoso, E; Jaraíz-Rodríguez, M; Medina, J M

    2016-05-26

    Connexin43 (Cx43) as a building block of gap junction channels and hemichannels exerts important functions in astrocytes. When these cells acquire a malignant phenotype Cx43 protein but not mRNA levels are downregulated, being negligible in high-grade astrocytoma or glioblastoma multiforme, the most common and deadliest of malignant primary brain tumors in adults. Some microRNAs associated to glioma target Cx43 and could explain the lack of correlation between mRNA and protein levels of Cx43 found in some high-grade astrocytomas. More importantly, these microRNAs could be a promising therapeutic target. A great number of studies have confirmed the relationship between cancer and connexins that was proposed by Loewenstein more than 40years ago, but these studies have also revealed that this is a very complex relationship. Indeed, restoring Cx43 to glioma cells reduces their rate of proliferation and their tumorigenicity but this tumor suppressor effect could be counterbalanced by its effects on invasiveness, adhesion and migration. The mechanisms underlying these effects suggest the participation of a great variety of proteins that bind to different regions of Cx43. The present review focuses on an intrinsically disordered region of the C-terminal domain of Cx43 in which converges the interaction of several proteins, including the proto-oncogene Src. We summarize data that indicate that Cx43-Src interaction inhibits the oncogenic activity of Src and promotes a conformational change in the structure of Cx43 that allosterically modifies the binding to other important signaling proteins. As a consequence, crucial cell functions, such as proliferation or migration, could be strongly affected. We propose that the knowledge of the structural basis of the antitumorigenic effect of Cx43 on astrocytomas could help to design new therapies against this incurable disease.

  20. Connexin 43 stabilizes astrocytes in a stroke-like milieu to facilitate neuronal recovery

    PubMed Central

    Wu, Le-yu; Yu, Xue-li; Feng, Lin-yin

    2015-01-01

    Aim: Connexin 43 (Cx43) is a member of connexin family mainly expressed in astrocytes, which forms gap junctions and hemichannels and maintains the normal shape and function of astrocytes. In this study we investigated the role of Cx43 in astrocytes in facilitating neuronal recovery during ischemic stroke. Methods: Primary culture of astrocytes or a mixed culture of astrocytes and cortical neurons was subjected to oxygen glucose deprivation and reperfusion (OGD/R). The expression of Cx43 and Ephrin-A4 in astrocytes was detected using immunocytochemical staining and Western blot assays. Intercellular Ca2+ concentration was determined with Fluo-4 AM fluorescent staining. Middle cerebral artery occlusion (MCAO) model rats were used for in vivo studies. Results: OGD/R treatment of cultured astrocytes caused a decrement of Cx43 expression and translocation of Cx43 from cell membrane to cytoplasm, accompanied by cell retraction. Furthermore, OGD/R increased intracellular Ca2+ concentration, activated CaMKII/CREB pathways and upregulated expression of Ephrin-A4 in the astrocytes. All these changes in OGD/R-treated astrocytes were alleviated by overexpression of Cx43. In the cortical neurons cultured with astrocytes, OGD/R inhibited the neurite growth, whereas overexpression of Cx43 or knockdown of Ephrin-A4 in astrocytes restored the neurite growth. In MCAO model rats, neuronal recovery was found to be correlated with the recuperation of Cx43 and Ephrin-A4 in astrocytes. Conclusion: Cx43 can stabilize astrocytes and facilitate the resistance to the deleterious effects of a stroke-like milieu and promote neuronal recovery. PMID:26095039

  1. Proteomic Analysis of Connexin 43 Reveals Novel Interactors Related to Osteoarthritis*

    PubMed Central

    Gago-Fuentes, Raquel; Fernández-Puente, Patricia; Megias, Diego; Carpintero-Fernández, Paula; Mateos, Jesus; Acea, Benigno; Fonseca, Eduardo; Blanco, Francisco Javier; Mayan, Maria Dolores

    2015-01-01

    We have previously reported that articular chondrocytes in tissue contain long cytoplasmic arms that physically connect two distant cells. Cell-to-cell communication occurs through connexin channels termed Gap Junction (GJ) channels, which achieve direct cellular communication by allowing the intercellular exchange of ions, small RNAs, nutrients, and second messengers. The Cx43 protein is overexpressed in several human diseases and inflammation processes and in articular cartilage from patients with osteoarthritis (OA). An increase in the level of Cx43 is known to alter gene expression, cell signaling, growth, and cell proliferation. The interaction of proteins with the C-terminal tail of connexin 43 (Cx43) directly modulates GJ-dependent and -independent functions. Here, we describe the isolation of Cx43 complexes using mild extraction conditions and immunoaffinity purification. Cx43 complexes were extracted from human primary articular chondrocytes isolated from healthy donors and patients with OA. The proteomic content of the native complexes was determined using LC-MS/MS, and protein associations with Cx43 were validated using Western blot and immunolocalization experiments. We identified >100 Cx43-associated proteins including previously uncharacterized proteins related to nucleolar functions, RNA transport, and translation. We also identified several proteins involved in human diseases, cartilage structure, and OA as novel functional Cx43 interactors, which emphasized the importance of Cx43 in the normal physiology and structural and functional integrity of chondrocytes and articular cartilage. Gene Ontology (GO) terms of the proteins identified in the OA samples showed an enrichment of Cx43-interactors related to cell adhesion, calmodulin binding, the nucleolus, and the cytoskeleton in OA samples compared with healthy samples. However, the mitochondrial proteins SOD2 and ATP5J2 were identified only in samples from healthy donors. The identification of Cx43

  2. Lipopolysaccharide Induces Degradation of Connexin43 in Rat Astrocytes via the Ubiquitin-Proteasome Proteolytic Pathway

    PubMed Central

    Liao, Chih-Kai; Jeng, Chung-Jiuan; Wang, Hwai-Shi; Wang, Shu-Huei; Wu, Jiahn-Chun

    2013-01-01

    The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway. PMID:24236122

  3. Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression.

    PubMed

    Moffitt, Julia A; Henry, Matthew K; Welliver, Kathryn C; Jepson, Amanda J; Garnett, Emily R

    2013-03-01

    Hindlimb unloading (HU) is a well-established animal model of cardiovascular deconditioning. Previous data indicate that HU results in cardiac sympathovagal imbalance. It is well established that cardiac sympathovagal imbalance increases the risk for developing cardiac arrhythmias. The cardiac gap junction protein connexin 43 (Cx43) is predominately expressed in the left ventricle (LV) and ensures efficient cell-to-cell electrical coupling. In the current study we wanted to test the hypothesis that HU would result in increased predisposition to cardiac arrhythmias and alter the expression and/or phosphorylation of LV-Cx43. Electrocardiographic data using implantable telemetry were obtained over a 10- to 14-day HU or casted control (CC) condition and in response to a sympathetic stressor using isoproterenol administration and brief restraint. The arrhythmic burden was calculated using a modified scoring system to quantify spontaneous and provoked arrhythmias. In addition, Western blot analysis was used to measure LV-Cx43 expression in lysates probed with antibodies directed against the total and an unphosphorylated form of Cx43 in CC and HU rats. HU resulted in a significantly greater total arrhythmic burden during the sympathetic stressor with significantly more ventricular arrhythmias occurring. In addition, there was increased expression of total LV-Cx43 observed with no difference in the expression of unphosphorylated LV-Cx43. Specifically, the increased expression of LV-Cx43 was consistent with the phosphorylated form. These data taken together indicate that cardiovascular deconditioning produced through HU results in increased predisposition to cardiac arrhythmias and increased expression of phosphorylated LV-Cx43.

  4. Effects of ischemic preconditioning on ischemia/reperfusion-induced arrhythmias by upregulatation of connexin 43 expression

    PubMed Central

    2011-01-01

    Background The susceptibility of hypertrophied myocardium to ischemia-reperfusion injury is associated with increased risk of postoperative arrhythmias. We investigate the effects of ischemic preconditioning (IP) on post-ischemic reperfusion arrhythmias in hypertrophic rabbit hearts. Methods Thirty-three rabbit models of myocardial hypertrophy were randomly divided into three groups of 11 each: non-ischemia-reperfusion group (group A), ischemia-reperfusion group (group B), and ischemic preconditioning group (group C). Another ten healthy rabbits with normal myocardium served as the healthy control group. Rabbit models of myocardial hypertrophy were induced by abdominal aortic banding. Surface electrocardiogram (ECG) was recorded and Curtis-Ravingerova score was used for arrhythmia quantification. Connexin 43 (Cx43) expression was assessed by immunohistochemistry. Results Ratios of heart weight to body weight and left ventricular weight to body weight increase significantly in the three groups compared with the healthy control group (p < 0.05). Arrhythmia incidence in group C is significantly lower than group B (p < 0.05). Curtis-Ravingerova score in group C is lower than group B (p < 0.05). Cx43 expression area in group A is smaller by comparison with the healthy control group (p < 0.05). Cx43 expression area and fluorescence intensity in group B are reduced by 60.9% and 23.9%, respectively, compared with group A (p < 0.05). In group C, Cx43 expression area increases by 32.5% compared with group B (p < 0.05), and decreases by 54.8% compared with group A (p < 0.05). Conclusions The incidence of ischemia/reperfusion-induced arrhythmias in hypertrophic rabbit hearts decreases after IP, which plays an important protecting role on the electrophysiology of hypertrophied myocardium by up-regulating the expression of Cx43. PMID:21635761

  5. Phosphorylation and subcellular distribution of connexin43 in normal and stressed cells.

    PubMed

    Leykauf, Kerstin; Dürst, Matthias; Alonso, Angel

    2003-01-01

    We have developed polyclonal antibodies (SA226P) to a peptide of the human connexin43 (Cx43) protein between amino acids 271 and 288 containing phosphorylated S279 and S282. Antibodies specific for the phosphorylated form of the peptide were isolated by double immunoaffinity chromatography and were characterised using proteins of the cell line WB-F344, known to contain large amounts of Cx43. SA226P recognises specifically the slowest migrating Cx43 band in immunoblots of proteins isolated from untreated cells. In immunofluorescence experiments SA226P scarcely stains the plasma membrane in untreated cells in contrast to a commercial antibody recognising all isoforms of the Cx43 protein. EGF or stress treatment of the cells results in a rapid increase in the phosphorylated forms of Cx43 as revealed by immunoblotting. Immunofluorescence experiments reveal that both phosphorylated and non-phosphorylated Cx43 could be found at the plasma membrane. Whether phosphorylation of S279/S282 takes place before or after incorporation of Cx43 into the membranes is so far unknown. More interestingly, confocal microscopy using our antibodies and a commercial antibody recognising all isoforms of Cx43 shows the coexistence of differentially phosphorylated forms of the protein at the plasma membrane. Our results indicate that MAP kinases erk1/2 are mainly responsible for this phosphorylation, as already published. Nevertheless, treatment of the cells with anisomycin, known to activate stress kinase p38 but not erk1/2, also results in a weak but reproducible Cx43 phosphorylation. PMID:12483281

  6. Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines

    PubMed Central

    Moinfar, Zahra; Dambach, Hannes; Schoenebeck, Bodo; Förster, Eckart; Prochnow, Nora; Faustmann, Pedro Michael

    2016-01-01

    Introduction Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43) and estrogen receptors (ERs). Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2) on Cx43 expression in two glioma cell lines with variable native expression of Cx43. Materials and Methods F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERβ and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique. Results E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERβ was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERβ in C6 cultures, while it decreased ERα expression in F98 glioma cells. Discussion These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma. PMID:26919293

  7. Gap-junction disassembly and connexin 43 dephosphorylation induced by 18 beta-glycyrrhetinic acid.

    PubMed

    Guan, X; Wilson, S; Schlender, K K; Ruch, R J

    1996-07-01

    Gap-junction channels connect the interiors of adjacent cells and can be arranged into aggregates or plaques consisting of hundreds to thousands of channel particles. The mechanism of channel aggregation into plaques and whether plaques can disaggregate are not known. Many carcinogenic and tumor-promoting chemicals have been identified that inhibit cell-cell gap-junctional coupling. Here, we provide morphological evidence that 18 beta-glycyrrhetinic acid (18 beta-GA), a saponin isolated from licorice root that is an inhibitor of gap-junctional communication, caused the disassembly of gap-junction plaques in WB-F344 rat liver epithelial cells. This effect was dose (5-40 microM) and time dependent (1-4 h treatment). Gap-junction channels in WB-F344 cells are comprised of connexin 43 (Cx43), and the protein is phosphorylated to a species known as Cx43-P2 coincident with the assembly of channels into plaques. Consistent with this, the disassembly of plaques induced by 18 beta-GA was correlated with decreases in Cx43-P2 levels and increases in nonphosphorylated Cx43. Biochemical evidence indicated that these changes in the P2 and NP forms of Cx43 represented 18 beta-GA-induced dephosphorylation of Cx43-P2 and not its degradation or the inhibition of Cx43-NP phosphorylation. Okadaic acid and calyculin A, which are inhibitors of type 1 and type 2A protein phosphatases, prevented the dephosphorylation of Cx43, suggesting that one or both of these phosphatases were involved in Cx43 dephosphorylation. These data indicate that 18 beta-GA causes type 1 or type 2A protein phosphatase-mediated Cx43 dephosphorylation coincident with the disassembly of gap-junction plaques.

  8. c-Src Kinase Inhibition Reduces Arrhythmia Inducibility and Connexin43 Dysregulation after Myocardial Infarction

    PubMed Central

    Rutledge, Cody A.; Ng, Fu Siong; Sulkin, Matthew S.; Greener, Ian D.; Sergeyenko, Artem M.; Liu, Hong; Gemel, Joanna; Beyer, Eric C.; Sovari, Ali A.; Efimov, Igor R.; Dudley, Samuel C.

    2014-01-01

    Objectives The aim of this study was to evaluate the role of c-Src inhibition on connexin43 (Cx43) regulation in a mouse model of myocardial infarction (MI). Background MI is associated with decreased expression of Cx43, the principal gap junction protein responsible for propagating current in ventricles. Activated c-Src has been linked to Cx43 dysregulation. Methods MI was induced in 12-week-old mice by coronary artery occlusion. MI mice were treated with c-Src inhibitors (PP1 or AZD0530), PP3 (an inactive analogue of PP1), or saline. Treated hearts were compared to sham mice by echocardiography, optical mapping, telemetry ECG monitoring, and inducibility studies. Tissues were collected for immunoblotting, quantitative PCR, and immunohistochemistry. Results Active c-Src was elevated in PP3-treated MI mice compared to sham at the scar border (280%, p=0.003) and distal ventricle (346%, p=0.013). PP1 treatment restored active c-Src to sham levels at the scar border (86%, p=0.95) and distal ventricle (94%, p=1.0). PP1 raised Cx43 expression by 69% in the scar border (p=0.048) and by 73% in distal ventricle (p=0.043) compared to PP3 mice. PP1-treated mice had restored conduction velocity at the scar border (PP3: 32 cm/s, PP1: 41 cm/s, p < 0.05) and lower arrhythmic inducibility (PP3: 71%, PP1: 35%, p < 0.05) than PP3 mice. PP1 did not change infarct size, ECG pattern, or cardiac function. AZD0530 treatment demonstrated restoration of Cx43 comparable to PP1. Conclusions c-Src inhibition improved Cx43 levels and conduction velocity and lowered arrhythmia inducibility after MI, suggesting a new approach for arrhythmia reduction following MI. PMID:24361364

  9. Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

    NASA Technical Reports Server (NTRS)

    Komarova, S. V.; Ataullakhanov, F. I.; Globus, R. K.

    2000-01-01

    To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

  10. The molecular role of connexin 43 in human trophoblast cell fusion.

    PubMed

    Dunk, Caroline E; Gellhaus, Alexandra; Drewlo, Sascha; Baczyk, Dora; Pötgens, Andy J G; Winterhager, Elke; Kingdom, John C P; Lye, Steven J

    2012-04-01

    Connexin expression and gap junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43)/gap junction A1 (GJA1) are required for cytotrophoblast fusion into the syncytium, the outer functional layer of the human placenta. Cx43 also impacts intracellular signaling through protein-protein interactions. The transcription factor GCM1 and its downstream target ERVW-1/SYNCYTIN-1 are key players in trophoblast fusion and exert their actions through the ERVW-1 receptor SLC1A5/ASCT-2/RDR/ATB(0). To investigate the molecular role of the Cx43 protein and its interaction with this fusogenic pathway, we utilized stable Cx43-transfected cell lines established from the choriocarcinoma cell line Jeg3: wild-type Jeg3, alphahCG/Cx43 (constitutive Cx43 expression), JpUHD/Cx43 (doxycyclin-inducible Cx43 expression), or JpUHD/trCx43 (doxycyclin-inducible Cx43 carboxyterminal deleted). We hypothesized that truncation of Cx43 at its C-terminus would inhibit trophoblast fusion and protein interaction with either ERVW-1 or SLC1A5. In the alphahCG/Cx43 and JpUHD/Cx43 lines, stimulation with cAMP caused 1) increase in GJA1 mRNA levels, 2) increase in percentage of fused cells, and 3) downregulation of SLC1A5 expression. Cell fusion was inhibited by GJIC blockade using carbenoxylone. Neither Jeg3, which express low levels of Cx43, nor the JpUHD/trCx43 cell line demonstrated cell fusion or downregulation of SLC1A5. However, GCM1 and ERVW-1 mRNAs were upregulated by cAMP treatment in both Jeg3 and all Cx43 cell lines. Silencing of GCM1 prevented the induction of GJA1 mRNA by forskolin in BeWo choriocarcinoma cells, demonstrating that GCM1 is upstream of Cx43. All cell lines and first-trimester villous explants also demonstrated coimmunoprecipitation of SLC1A5 and phosphorylated Cx43. Importantly, SLC1A5 and Cx43 gap junction plaques colocalized in situ to areas of fusing cytotrophoblast, as demonstrated by the loss of E-cadherin staining in the plasma membrane in first

  11. Ginkgo suppresses atherosclerosis through downregulating the expression of connexin 43 in rabbits

    PubMed Central

    Wang, Xin; Gong, Hui; Shi, Yi Jun; Zou, Yunzeng

    2013-01-01

    Introduction Ginkgo biloba extract (GBE) EGb761 is widely used for cardiovascular prevention. Here, we investigated the effects of GBE on atherosclerotic lesion development in rabbits with a high-fat diet. Material and methods Forty New Zealand white male rabbits were randomly divided into four groups. The first two were the normal diet group (C) and the high-fat group (HF). The remaining two groups were those who received a high cholesterol diet supplemented with either the standard drug (simvastatin 2 mg/kg/day) or GBE (3 mg/kg/day). At 12 weeks, histopathological and chemical analyses were performed. Results Plasma lipid measurement showed that GBE inhibited high-fat diet-induced increase of serum triglyceride (TG), total cholesterol (TC), and low density lipoprotein cholesterol (LDL-C) by 59.1% (0.9 ±0.2 4 mmol/l vs. 2.2 ±0.4 mmol/l), 18.2% (31.1 ±1.4 mmol/l vs. 38.0 ±0.4 mmol/l) and 15% (28.9 ±1.3 mmol/l vs. 34.0±1.0 mmol/l), respectively, at 12 weeks (p < 0.01). The en face Sudan IV-positive lesion area of the aorta in the GBE group (51.7 ±3.1%) was significantly lower compared with that in the HF group (88.2 ±2.2%; p < 0.01). The mean atherosclerotic lesion area of the GBE group was reduced by 53.2% compared with the HF group (p < 0.01). Immunohistochemistry and western blot analysis showed that GBE markedly suppressed high-fat diet-induced upregulation of connexin 43 (Cx43) in rabbits (p < 0.01). Conclusions Thus, our study revealed that GBE prevented atherosclerosis progress through modulating plasma lipid, suppressing atherosclerotic lesion development, and attenuating the expression of Cx43 protein. PMID:23671447

  12. Deficiency of cyclase-associated protein 2 promotes arrhythmias associated with connexin43 maldistribution and fibrosis

    PubMed Central

    Peche, Vivek Shahaji; Linhart, Markus; Nickenig, Georg; Noegel, Angelika Anna; Schrickel, Jan Wilko

    2016-01-01

    Introduction Cyclase-associated protein 2 (CAP2) plays a major role in regulating the actin cytoskeleton. Since inactivation of CAP2 in a mouse model by a gene trap approach (Cap2gt/gt) results in cardiomyopathy and increased mortality, we hypothesized that CAP2 has a major impact on arrhythmias and electrophysiological parameters. Material and methods We performed long-term-ECG recordings in transgenic CAP2 deficient mice (C57BL/6) to detect spontaneous arrhythmias. In vivo electrophysiological studies by right heart catheterization and ex vivo epicardial mapping were used to analyze electrophysiological parameters, the inducibility of arrhythmias, and conduction velocities. Expression and distribution of cardiac connexins and the amount of cardiac fibrosis were evaluated. Results Spontaneous ventricular arrhythmias could be detected in Cap2gt/gt during the long-term-ECG recording. Cap2gt/gt showed marked conduction delays at atrial and ventricular levels, including a reduced heart rate (421.0 ±40.6 bpm vs. 450.8 ±27.9 bpm; p < 0.01), and prolongations of PQ (46.3 ±4.1 ms vs. 38.6 ±6.5 ms; p < 0.01), QRS (16.2 ±2.6 ms vs. 12.6 ±1.4 ms; p < 0.01), and QTc interval (55.8 ±6.0 ms vs. 45.2 ±3.3 ms; p = 0.02) in comparison to wild type mice. The PQ prolongation was due to an infra-Hisian conduction delay (HV: 9.7 ±2.1 ms vs. 6.5 ±3.1 ms; p = 0.02). The inducibility of ventricular tachycardias during the electrophysiological studies was significantly elevated in the mutant mice (inducible animals: 88% vs. 33%; p = 0.04). Cap2gt/gt showed more abnormal distribution of connexin43 compared to WT (23.0 ±4.7% vs. 2.9 ±0.8%; p < 0.01). Myocardial fibrosis was elevated in Cap2gt/gt hearts (9.1 ±6.7% vs. 5.5 ±3.3%; p < 0.01). Conclusions Loss of CAP2 results in marked electrophysiological disturbances including impaired sinus node function, conduction delays, and susceptibility to malignant arrhythmias. Structural changes in Cap2gt/gt are associated with

  13. Myoblast proliferation and syncytial fusion both depend on connexin43 function in transfected skeletal muscle primary cultures

    SciTech Connect

    Gorbe, Aniko; Krenacs, Tibor; Cook, Jeremy E.; Becker, David L. . E-mail: d.becker@ucl.ac.uk

    2007-04-01

    Muscles are formed by fusion of individual postmitotic myoblasts to form multinucleated syncytial myotubes. The process requires a well-coordinated transition from proliferation, through migratory alignment and cycle exit, to breakdown of apposed membranes. Connexin43 protein and cell-cycle inhibitor levels are correlated, and gap junction blockers can delay muscle regeneration, so a coordinating role for gap junctions has been proposed. Here, wild-type and dominant-negative connexin43 variants (wtCx43, dnCx43) were introduced into rat myoblasts in primary culture through pIRES-eGFP constructs that made transfected cells fluoresce. GFP-positive cells and vitally-stained nuclei were counted on successive days to reveal differences in proliferation, and myotubes were counted to reveal differences in fusion. Individual transfected cells were injected with Cascade Blue, which permeates gap junctions, mixed with FITC-dextran, which requires cytoplasmic continuity to enter neighbouring cells. Myoblasts transfected with wtCx43 showed more gap-junctional coupling than GFP-only controls, began fusion sooner as judged by the incidence of cytoplasmic coupling, and formed more myotubes. Myoblasts transfected with dnCx43 remained proliferative for longer than either GFP-only or wtCx43 myoblasts, showed less coupling, and underwent little fusion into myotubes. These results highlight the critical role of gap-junctional coupling in myotube formation.

  14. Effects of Electroacupuncture on Interstitial Cells of Cajal (ICC) Ultrastructure and Connexin 43 Protein Expression in the Gastrointestinal Tract of Functional Dyspepsia (FD) Rats.

    PubMed

    Zhang, Guoshan; Xie, Shen; Hu, Wei; Liu, Yuer; Liu, Mailan; Liu, Mi; Chang, Xiaorong

    2016-01-01

    BACKGROUND Gastrointestinal motility disorder is the main clinical manifestation in functional dyspepsia (FD) patients. Electroacupuncture is effective in improving gastrointestinal motility disorder in FD; however, the underlying mechanism remains unclear. It has been demonstrated that interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal tract, and the pacemaker potential is transmitted to nearby cells through gap junctions between ICC or ICC and the smooth muscle. Therefore, this study aimed to assess the effects of electroacupuncture on ICC ultrastructure and expression of the gap junction protein connexin 43 (Cx43) in FD rats. MATERIAL AND METHODS The animals were randomized into 3 groups: control, model, and electroacupuncture. Electroacupuncture was applied at Zusanli (ST36) in the electroacupuncture group daily for 10 days, while no electroacupuncture was applied to model group animals. RESULTS Ultrastructure of ICC recovered normally in gastric antrum and small intestine specimens was improved, with Cx43 expression levels in these tissues significantly increased in the electroacupuncture group compared with the model group. CONCLUSIONS These findings indicated that electroacupuncture is effective in alleviating ICC damage and reduces Cx43 levels in FD rats, and suggest that ICC and Cx43 are involved in electroacupuncture treatment in rats with FD to improve gastrointestinal motility disorders. PMID:27297942

  15. Effects of Electroacupuncture on Interstitial Cells of Cajal (ICC) Ultrastructure and Connexin 43 Protein Expression in the Gastrointestinal Tract of Functional Dyspepsia (FD) Rats.

    PubMed

    Zhang, Guoshan; Xie, Shen; Hu, Wei; Liu, Yuer; Liu, Mailan; Liu, Mi; Chang, Xiaorong

    2016-01-01

    BACKGROUND Gastrointestinal motility disorder is the main clinical manifestation in functional dyspepsia (FD) patients. Electroacupuncture is effective in improving gastrointestinal motility disorder in FD; however, the underlying mechanism remains unclear. It has been demonstrated that interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal tract, and the pacemaker potential is transmitted to nearby cells through gap junctions between ICC or ICC and the smooth muscle. Therefore, this study aimed to assess the effects of electroacupuncture on ICC ultrastructure and expression of the gap junction protein connexin 43 (Cx43) in FD rats. MATERIAL AND METHODS The animals were randomized into 3 groups: control, model, and electroacupuncture. Electroacupuncture was applied at Zusanli (ST36) in the electroacupuncture group daily for 10 days, while no electroacupuncture was applied to model group animals. RESULTS Ultrastructure of ICC recovered normally in gastric antrum and small intestine specimens was improved, with Cx43 expression levels in these tissues significantly increased in the electroacupuncture group compared with the model group. CONCLUSIONS These findings indicated that electroacupuncture is effective in alleviating ICC damage and reduces Cx43 levels in FD rats, and suggest that ICC and Cx43 are involved in electroacupuncture treatment in rats with FD to improve gastrointestinal motility disorders.

  16. Effects of Electroacupuncture on Interstitial Cells of Cajal (ICC) Ultrastructure and Connexin 43 Protein Expression in the Gastrointestinal Tract of Functional Dyspepsia (FD) Rats

    PubMed Central

    Zhang, Guoshan; Xie, Shen; Hu, Wei; Liu, Yuer; Liu, Mailan; Liu, Mi; Chang, Xiaorong

    2016-01-01

    Background Gastrointestinal motility disorder is the main clinical manifestation in functional dyspepsia (FD) patients. Electroacupuncture is effective in improving gastrointestinal motility disorder in FD; however, the underlying mechanism remains unclear. It has been demonstrated that interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal tract, and the pacemaker potential is transmitted to nearby cells through gap junctions between ICC or ICC and the smooth muscle. Therefore, this study aimed to assess the effects of electroacupuncture on ICC ultrastructure and expression of the gap junction protein connexin 43 (Cx43) in FD rats. Material/Methods The animals were randomized into 3 groups: control, model, and electroacupuncture. Electroacupuncture was applied at Zusanli (ST36) in the electroacupuncture group daily for 10 days, while no electroacupuncture was applied to model group animals. Results Ultrastructure of ICC recovered normally in gastric antrum and small intestine specimens was improved, with Cx43 expression levels in these tissues significantly increased in the electroacupuncture group compared with the model group. Conclusions These findings indicated that electroacupuncture is effective in alleviating ICC damage and reduces Cx43 levels in FD rats, and suggest that ICC and Cx43 are involved in electroacupuncture treatment in rats with FD to improve gastrointestinal motility disorders. PMID:27297942

  17. Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation.

    PubMed

    Yamada, Aya; Futagi, Masaharu; Fukumoto, Emiko; Saito, Kan; Yoshizaki, Keigo; Ishikawa, Masaki; Arakaki, Makiko; Hino, Ryoko; Sugawara, Yu; Ishikawa, Momoko; Naruse, Masahiro; Miyazaki, Kanako; Nakamura, Takashi; Fukumoto, Satoshi

    2016-01-01

    Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43(-/-) salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43(-/-) samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43(-/-) phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.

  18. Effect of lysophosphatidic acid on the immune inflammatory response and the connexin 43 protein in myocardial infarction

    PubMed Central

    ZHANG, DUODUO; ZHANG, YAN; ZHAO, CHUNYAN; ZHANG, WENJIE; SHAO, GUOGUANG; ZHANG, HONG

    2016-01-01

    Lysophosphatidic acid (LPA) is an intermediate product of membrane phospholipid metabolism. Recently, LPA has gained attention for its involvement in the pathological processes of certain cardiovascular diseases. The aim of the present study was to clarify the association between the effect of LPA and the immune inflammatory response, and to investigate the effects of LPA on the protein expression levels of connexin 43 during myocardial infarction. Surface electrocardiograms of myocardial infarction rats and isolated rat heart tissue samples were obtained in order to determine the effect of LPA on the incidence of arrhythmia in rats that exhibited changes in immune status. The results demonstrated that the incidence of arrhythmia decreased when the rat immune systems were suppressed, and the incidence of arrhythmia increased when the rat immune systems were enhanced. The concentration levels of tumor necrosis factor (TNF)-α were determined by ELISA, and the results demonstrated that LPA induced T lymphocyte synthesis and TNF-α release. Using a patch-clamp technique, LPA was shown to increase the current amplitude of the voltage-dependent potassium channels (Kv) and calcium-activated potassium channels (KCa) in Jurkat T cells. The protein expression of connexin 43 (Cx43) was determined by immunohistochemical staining. The results indicated that LPA caused the degradation of Cx43 and decreased the expression of Cx43. This effect was associated with the immune status of the rats. There was a further decrease in Cx43 expression in the rats of the immune-enhanced group. To the best of our knowledge, these results provide the first evidence that LPA causes arrhythmia through the regulation of immune inflammatory cells and the decrease of Cx43 protein expression. The present study provided an experimental basis for the treatment of arrhythmia and may guide clinical care. PMID:27168781

  19. Altered Connexin 43 and Connexin 45 protein expression in the heart as a function of social and environmental stress in the prairie vole.

    PubMed

    Grippo, Angela J; Moffitt, Julia A; Henry, Matthew K; Firkins, Rachel; Senkler, Jonathan; McNeal, Neal; Wardwell, Joshua; Scotti, Melissa-Ann L; Dotson, Ashley; Schultz, Rachel

    2015-01-01

    Exposure to social and environmental stressors may influence behavior as well as autonomic and cardiovascular regulation, potentially leading to depressive disorders and cardiac dysfunction including elevated sympathetic drive, reduced parasympathetic function, and ventricular arrhythmias. The cellular mechanisms that underlie these interactions are not well understood. One mechanism may involve alterations in the expression of Connexin43 (Cx43) and Connexin45 (Cx45), gap junction proteins in the heart that play an important role in ensuring efficient cell-to-cell coupling and the maintenance of cardiac rhythmicity. The present study investigated the hypothesis that long-term social isolation, combined with mild environmental stressors, would produce both depressive behaviors and altered Cx43 and Cx45 expression in the left ventricle of prairie voles - a socially monogamous rodent model. Adult, female prairie voles were exposed to either social isolation (n = 22) or control (paired, n = 23) conditions (4 weeks), alone or in combination with chronic mild stress (CMS) (1 week). Social isolation, versus paired control conditions, produced significantly (p < 0.05) increased depressive behaviors in a 5-min forced swim test, and CMS exacerbated (p < 0.05) these behaviors. Social isolation (alone) reduced (p < 0.05) total Cx43 expression in the left ventricle; whereas CMS (but not isolation) increased (p < 0.05) total Cx45 expression and reduced (p < 0.05) the Cx43/Cx45 ratio, measured via Western blot analysis. The present findings provide insight into potential cellular mechanisms underlying altered cardiac rhythmicity associated with social and environmental stress in the prairie vole.

  20. Altered Connexin 43 and Connexin 45 Protein Expression in the Heart as a Function of Social and Environmental Stress in the Prairie Vole

    PubMed Central

    Grippo, Angela J.; Moffitt, Julia A.; Henry, Matthew K.; Firkins, Rachel; Senkler, Jonathan; McNeal, Neal; Wardwell, Joshua; Scotti, Melissa-Ann L.; Dotson, Ashley; Schultz, Rachel

    2015-01-01

    Exposure to social and environmental stressors may influence behavior as well as autonomic and cardiovascular regulation, potentially leading to depressive disorders and cardiac dysfunction including elevated sympathetic drive, reduced parasympathetic function, and ventricular arrhythmias. The cellular mechanisms that underlie these interactions are not well understood. One mechanism may involve alterations in the expression of Connexin43 (Cx43) and Connexin45 (Cx45), gap junction proteins in the heart that play an important role in ensuring efficient cell-to-cell coupling and the maintenance of cardiac rhythmicity. The present study investigated the hypothesis that long-term social isolation, combined with mild environmental stressors, would produce both depressive behaviors and altered Cx43 and Cx45 expression in the left ventricle of prairie voles – a socially monogamous rodent model. Adult, female prairie voles were exposed to either social isolation (n=22) or control (paired, n=23) conditions (4 weeks), alone or in combination with chronic mild stress (1 week). Social isolation, versus paired control conditions, produced significantly (P < 0.05) increased depressive behaviors in a 5-min forced swim test, and chronic mild stress exacerbated (P < 0.05) these behaviors. Social isolation (alone) reduced (P < 0.05) total Cx43 expression in the left ventricle; whereas chronic mild stress (but not isolation) increased (P < 0.05) total Cx45 expression and reduced (P < 0.05) the Cx43/Cx45 ratio, measured via Western blot analysis. The present findings provide insight into potential cellular mechanisms underlying altered cardiac rhythmicity associated with social and environmental stress in the prairie vole. PMID:25338193

  1. Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion.

    PubMed

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Gentilini, María Virginia; Velásquez, Lis Noelia; Fossati, Carlos Alberto; Giambartolomei, Guillermo Hernán; Delpino, María Victoria

    2015-10-12

    Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-β, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage.

  2. Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion

    PubMed Central

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Gentilini, María Virginia; Velásquez, Lis Noelia; Fossati, Carlos Alberto; Giambartolomei, Guillermo Hernán

    2015-01-01

    Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-β, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage. PMID:26459511

  3. Intercellular communication within the rat anterior pituitary gland: X. Immunohistocytochemistry of S-100 and connexin 43 of folliculo-stellate cells in the rat anterior pituitary gland.

    PubMed

    Shirasawa, Nobuyuki; Mabuchi, Yoshio; Sakuma, Eisuke; Horiuchi, Osamu; Yashiro, Takashi; Kikuchi, Motoshi; Hashimoto, Yasuo; Tsuruo, Yoshihiro; Herbert, Damon C; Soji, Tsuyoshi

    2004-05-01

    Since Rinehart and Farquhar reported the presence of agranulated cells in the anterior pituitary gland in 1953, the functions of the folliculo-stellate cell remain to be clarified. Intercellular junctions have been described in the monkey, rat, and teleost anterior pituitary glands, indicating the existence of cell-to-cell communication within the organ. We pointed to their possible role in the rapid dissemination of information through a complex interconnecting system of follicles involving gap junctions. The gap junctional/folliculo-stellate cellular network was essential in the maturation and regulation of the pituitary gland system such as the hypothalamic-pituitary-gonadal axis. It has been was shown that a network participated in the conduction of electrophysiological information over a long distance using the ion Ca(++), which propagates to other folliculo-stellate cells by signaling through gap junctions. Sixty-day-old male rats were used in this study for light microscopic immunohistochemistry of S-100 protein, type I collagen, and connexin 43, and for electron microscopy to observe the morphological relationships between the cellular networks of folliculo-stellate cells and granulated pituitary cells. Clusters of anti-S-100 protein-positive cells were clearly observed in a region of the hypophysis tentatively named the transition zone. Anti-S-100 protein-positive cells and their cytoplasmic processes were also present in the anterior lobe and assembled together to form follicular lumina. Type I collagen was clearly shown outlining the incomplete lobular or ductule-like structure making cell cords in the anterior pituitary gland. Numerous microvilli were present within the follicular lumen while around the lumina, junctional specializations including gap junctions were positive for the connexin 43 protein. A nonuniform distribution of the connexin 43-positive sites were observed. Small or dot-shaped positive sites were noted where two clusters of cells

  4. Oocyte-derived BMP15 but not GDF9 down-regulates connexin43 expression and decreases gap junction intercellular communication activity in immortalized human granulosa cells.

    PubMed

    Chang, Hsun-Ming; Cheng, Jung-Chien; Taylor, Elizabeth; Leung, Peter C K

    2014-05-01

    In the ovary, connexin-coupled gap junctions in granulosa cells play crucial roles in follicular and oocyte development as well as in corpus luteum formation. Our previous work has shown that theca cell-derived bone morphogenetic protein (BMP)4 and BMP7 decrease gap junction intercellular communication (GJIC) activity via the down-regulation of connexin43 (Cx43) expression in immortalized human granulosa cells. However, the effects of oocyte-derived growth factors on Cx43 expression remain to be elucidated. The present study was designed to investigate the effects of oocyte-derived growth differentiation factor (GDF)9 and BMP15 on the expression of Cx43 in a human granulosa cell line, SVOG. We also examined the effect relative to GJIC activity and investigated the potential mechanisms of action. In SVOG cells, treatment with BMP15 but not GDF9 significantly decreased Cx43 mRNA and protein levels and GJIC activity. These suppressive effects, along with the induction of Smad1/5/8 phosphorylation, were attenuated by co-treatment with a BMP type I receptor inhibitor, dorsomorphin. Furthermore, knockdown of the central component of the transforming growth factor-β superfamily signaling pathway, Smad4, using small interfering RNA reversed the suppressive effects of BMP15 on Cx43 expression and GJIC activity. The suppressive effects of BMP15 on Cx43 expression were further confirmed in primary human granulosa-lutein cells obtained from infertile patients undergoing an in vitro fertilization procedure. These findings suggest that oocyte-derived BMP15 decreases GJIC activity between human granulosa cells by down-regulating Cx43 expression, most likely via a Smad-dependent signaling pathway.

  5. Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption.

    PubMed

    Li, Nan; Mruk, Dolores D; Mok, Ka-Wai; Li, Michelle W M; Wong, Chris K C; Lee, Will M; Han, Daishu; Silvestrini, Bruno; Cheng, C Yan

    2016-04-01

    Earlier studies have shown that rats treated with an acute dose of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (adjudin, a male contraceptive under development) causes permanent infertility due to irreversible blood-testis barrier (BTB) disruption even though the population of undifferentiated spermatogonia remains similar to normal rat testes, because spermatogonia fail to differentiate into spermatocytes to enter meiosis. Since other studies have illustrated the significance of connexin 43 (Cx43)-based gap junction in maintaining the homeostasis of BTB in the rat testis and the phenotypes of Sertoli cell-conditional Cx43 knockout mice share many of the similarities of the adjudin-treated rats, we sought to examine if overexpression of Cx43 in these adjudin-treated rats would reseal the disrupted BTB and reinitiate spermatogenesis. A full-length Cx43 cloned into mammalian expression vector pCI-neo was used to transfect testes of adjudin-treated ratsversusempty vector. It was found that overexpression of Cx43 indeed resealed the Sertoli cell tight junction-permeability barrier based on a functionalin vivoassay in tubules displaying signs of meiosis as noted by the presence of round spermatids. Thus, these findings suggest that overexpression of Cx43 reinitiated spermatogenesis at least through the steps of meiosis to generate round spermatids in testes of rats treated with an acute dose of adjudin that led to aspermatogenesis. It was also noted that the round spermatids underwent eventual degeneration with the formation of multinucleated cells following Cx43 overexpression due to the failure of spermiogenesis because no elongating/elongated spermatids were detected in any of the tubules examined. The mechanism by which overexpression of Cx43 reboots meiosis and rescues BTB function was also examined. In summary, overexpression of Cx43 in the testis with aspermatogenesis reboots meiosis and reseals toxicant-induced BTB disruption, even though it fails to

  6. Effects of broad frequency vibration on cultured osteoblasts

    NASA Technical Reports Server (NTRS)

    Tanaka, Shigeo M.; Li, Jiliang; Duncan, Randall L.; Yokota, Hiroki; Burr, David B.; Turner, Charles H.

    2003-01-01

    Bone is subjected in vivo to both high amplitude, low frequency strain, incurred by locomotion, and to low amplitude, broad frequency strain. The biological effects of low amplitude, broad frequency strain are poorly understood. To evaluate the effects of low amplitude strains ranging in frequency from 0 to 50 Hz on osteoblastic function, we seeded MC3T3-E1 cells into collagen gels and applied the following loading protocols for 3 min per day for either 3 or 7 days: (1) sinusoidal strain at 3 Hz, with 0-3000 microstrain peak-to-peak followed by 0.33 s resting time, (2) "broad frequency vibration" of low amplitude strain (standard deviation of 300 microstrain) including frequency components from 0 to 50 Hz, and (3) sinusoidal strain combined with broad frequency vibration (S + V). The cells were harvested on day 4 or 8. We found that the S + V stimulation significantly repressed cell proliferation by day 8. Osteocalcin mRNA was up-regulated 2.6-fold after 7 days of S + V stimulation, and MMP-9 mRNA was elevated 1.3-fold after 3 days of vibration alone. Sinusoidal stimulation alone did not affect the cell responses. No differences due to loading were observed in alkaline phosphatase activity and in mRNA levels of type I collagen, osteopontin, connexin 43, MMPs-1A, -3, -13. These results suggest that osteoblasts are more sensitive to low amplitude, broad frequency strain, and this kind of strain could sensitize osteoblasts to high amplitude, low frequency strain. This suggestion implies a potential contribution of stochastic resonance to the mechanical sensitivity of osteoblasts. Copyright 2002 Elsevier Science Ltd.

  7. The B[a]P-increased intercellular communication via translocation of connexin-43 into gap junctions reduces apoptosis

    SciTech Connect

    Tekpli, X.; Rivedal, E.; Gorria, M.; Landvik, N.E.; Rissel, M.; Dimanche-Boitrel, M.-T.; Baffet, G.; Holme, J.A.; Lagadic-Gossmann, D.

    2010-01-15

    Gap junctions are channels in plasma membrane composed of proteins called connexins. These channels are organized in special domains between cells, and provide for direct gap junctional intercellular communication (GJIC), allowing diffusion of signalling molecules < 1 kD. GJIC regulates cell homeostasis and notably the balance between proliferation, cell cycle arrest, cell survival and apoptosis. Here, we have investigated benzo[a]pyrene (B[a]P) effects on GJIC and on the subcellular localization of the major protein of gap junction: connexin-43 (Cx43). Our results showed that B[a]P increased GJIC between mouse hepatoma Hepa1c1c7 cells via translocation of Cx43 from Golgi apparatus and lipid rafts into gap junction plaques. Interestingly, inhibition of GJIC by chlordane or small interference RNA directed against Cx43 enhanced B[a]P-induced apoptosis in Hepa1c1c7 cells. The increased apoptosis caused by inhibition of GJIC appeared to be mediated by ERK/MAPK pathway. It is suggested that B[a]P could induce transfer of cell survival signal or dilute cell death signal via regulation of ERK/MAPK through GJIC.

  8. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

    PubMed Central

    Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

    2016-01-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction. PMID:27436542

  9. Akt Phosphorylates Connexin43 on Ser373, a “Mode-1” Binding Site for 14-3-3

    PubMed Central

    PARK, DARREN J.; WALLICK, CHRISTOPHER J.; MARTYN, KENDRA D.; LAU, ALAN F.; JIN, CHENGSHI; WARN-CRAMER, BONNIE J.

    2009-01-01

    Connexin43 (Cx43) is a membrane-spanning protein that forms channels that bridge the gap between adjacent cells and this allows for the intercellular exchange of information. Cx43 is regulated by phosphorylation and by interacting proteins. “Mode-1” interaction with 14-3-3 requires phosphorylation of Ser373 on Cx43 (Park et al. 2006). Akt phosphorylates and targets a number of proteins to interactions with 14-3-3. Here we demonstrate that Akt phosphorylates Cx43 on Ser373 and Ser369; antibodies recognizing Akt-phosphorylated sites or phospho-Ser “mode-1” 14-3-3-binding sites recognize a protein from EGF-treated cells that migrates as Cx43, and GST-14-3-3 binds to Cx43 phosphorylated endogenously in EGF-treated cells. Confocal microscopy supports the co-localization of Cx43 with Akt and with 14-3-3 at the outer edges of gap junctional plaques. These data suggest that Akt could target Cx43 to an interaction with 14-3-3 that may play a role in the forward trafficking of Cx43 multimers and/or their incorporation into existing gap junctional plaques. PMID:18163231

  10. Topical Administration of a Connexin43-based peptide Augments Healing of Chronic Neuropathic Diabetic Foot Ulcers: A Multicenter, Randomized Trial

    PubMed Central

    Grek, Christina L.; Prasad, G.M.; Viswanathan, Vijay; Armstrong, David G.; Gourdie, Robert G.; Ghatnekar, Gautam S.

    2015-01-01

    Nonhealing neuropathic foot ulcers remain a significant problem in individuals with diabetes. The gap-junctional protein connexin43 (Cx43) has roles in dermal wound healing and targeting Cx43 signaling accelerates wound reepithelialization. In a prospective, randomized, multi-center clinical trial we evaluated the efficacy and safety of a peptide mimetic of the C-terminus of Cx43, ACT1, in accelerating the healing of chronic diabetic foot ulcers (DFUs) when incorporated into standard of care protocols. Adults with DFUs of at least four weeks duration were randomized to receive standard of care with or without topical application of ACT1. Primary outcome was mean percent ulcer reepithelialization and safety variables included incidence of treatment related adverse events and detection of ACT1 immunogenicity. ACT1 treatment was associated with a significantly greater reduction in mean percent ulcer area from baseline to 12 weeks (72.1% vs. 57.1%; p = 0.03). Analysis of incidence and median time-to-complete-ulcer closure revealed that ACT1 treatment was associated with a greater percentage of participants that reached 100% ulcer reepitheliazation and a reduced median time-to-complete-ulcer closure. No adverse events reported were treatment related, and ACT1 was not immunogenic. Treatment protocols that incorporate ACT1 may present a therapeutic strategy that safely augments the reepithelialization of chronic DFUs. PMID:25703647

  11. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

    NASA Astrophysics Data System (ADS)

    Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

    2016-07-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction.

  12. The Effect of a Connexin43-Based Peptide on the Healing of Chronic Venous Leg Ulcers: A Multicenter, Randomized Trial

    PubMed Central

    Ghatnekar, Gautam S; Grek, Christina L; Armstrong, David G; Desai, Sanjay C; Gourdie, Robert G

    2015-01-01

    The gap junction protein, connexin43 (Cx43), has critical roles in the inflammatory, edematous, and fibrotic processes following dermal injury and during wound healing, and is abnormally upregulated at the epidermal wound margins of venous leg ulcers (VLUs). Targeting Cx43 with ACT1, a peptide mimetic of the carboxyl-terminus of Cx43, accelerates fibroblast migration and proliferation, and wound reepithelialization. In a prospective, multicenter clinical trial conducted in India, adults with chronic VLUs were randomized to treatment with an ACT1 gel formulation plus conventional standard-of-care (SOC) protocols, involving maintaining wound moisture and four-layer compression bandage therapy, or SOC protocols alone. The primary end point was mean percent ulcer reepithelialization from baseline to 12 weeks. A significantly greater reduction in mean percent ulcer area from baseline to 12 weeks was associated with the incorporation of ACT1 therapy (79% (SD 50.4)) as compared with compression bandage therapy alone (36% (SD 179.8); P=0.02). Evaluation of secondary efficacy end points indicated a reduced median time to 50 and 100% ulcer reepithelialization for ACT1-treated ulcers. Incorporation of ACT1 in SOC protocols may represent a well-tolerated, highly effective therapeutic strategy that expedites chronic venous ulcer healing by treating the underlying ulcer pathophysiology through Cx43-mediated pathways. PMID:25072595

  13. AMPK Suppresses Connexin43 Expression in the Bladder and Ameliorates Voiding Dysfunction in Cyclophosphamide-induced Mouse Cystitis

    PubMed Central

    Zhang, Xiling; Yao, Jian; Gao, Kun; Chi, Yuan; Mitsui, Takahiko; Ihara, Tatsuya; Sawada, Norifumi; Kamiyama, Manabu; Fan, Jianglin; Takeda, Masayuki

    2016-01-01

    Bladder voiding dysfunction is closely related to local oxidation, inflammation, and enhanced channel activities. Given that the AMP-activated protein kinase (AMPK) has anti-oxidative, anti-inflammatory and channel-inhibiting properties, we examined whether and how AMPK affected bladder activity. AMPK activation in rat bladder smooth muscle cells (BSMCs) using three different AMPK agonists resulted in a decrease in connexin43 (Cx43) expression and function, which was associated with reduced CREB phosphorylation, Cx43 promoter activity and mRNA expression, but not Cx43 degradation. Downregulation of CREB with siRNA increased Cx43 expression. A functional analysis revealed that AMPK weakened BSMC contraction and bladder capacity. AMPK also counteracted the IL-1β- and TNFα-induced increase in Cx43 in BSMCs. In vivo administration of the AMPK agonist AICAR attenuated cyclophosphamide-initiated bladder oxidation, inflammation, Cx43 expression and voiding dysfunction. Further analysis comparing the responses of the wild-type (Cx43+/+) and heterozygous (Cx43+/−) Cx43 mice to cyclophosphamide revealed that the Cx43+/− mice retained a relatively normal micturition pattern compared to the Cx43+/+ mice. Taken together, our results indicate that AMPK inhibits Cx43 in BSMCs and improves bladder activity under pathological conditions. We propose that strategies that target AMPK can be developed as novel therapeutic approaches for treating bladder dysfunction. PMID:26806558

  14. Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers*

    PubMed Central

    Carnarius, Christian; Kreir, Mohamed; Krick, Marcel; Methfessel, Christoph; Moehrle, Volker; Valerius, Oliver; Brüggemann, Andrea; Steinem, Claudia; Fertig, Niels

    2012-01-01

    In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein. PMID:22139870

  15. Cardiac connexin-43 and PKC signaling in rats with altered thyroid status without and with omega-3 fatty acids intake.

    PubMed

    Szeiffová Bačová, B; Egan Beňová, T; Viczenczová, C; Soukup, T; Rauchová, H; Pavelka, S; Knezl, V; Barančík, M; Tribulová, N

    2016-09-19

    Thyroid hormones are powerful modulators of heart function and susceptibility to arrhythmias via both genomic and non-genomic actions. We aimed to explore expression of electrical coupling protein connexin-43 (Cx43) in the heart of rats with altered thyroid status and impact of omega-3 polyunsaturated fatty acids (omega-3) supplementation. Adult male Lewis rats were divided into following six groups: euthyroid controls, hyperthyroid (treated with T(3)) and hypothyroid (treated with methimazol) with or without six-weeks lasting supplementation with omega-3 (20 mg/100 g/day). Left and right ventricles, septum and atria were used for immunoblotting of Cx43 and protein kinase C (PKC). Total expression of Cx43 and its phosphorylated forms were significantly increased in all heart regions of hypothyroid rats compared to euthyroid controls. In contrast, the total levels of Cx43 and its functional phosphorylated forms were decreased in atria and left ventricle of hyperthyroid rats. In parallel, the expression of PKC epsilon that phosphorylates Cx43, at serine 368, was increased in hypothyroid but decreased in hyperthyroid rat hearts. Omega-3 intake did not significantly affect either Cx43 or PKC epsilon alterations. In conclusion, there is an inverse relationship between expression of cardiac Cx43 and the levels of circulating thyroid hormones. It appears that increased propensity of hyperthyroid while decreased of hypothyroid individuals to malignant arrhythmias may be in part attributed to the changes in myocardial Cx43.

  16. Mechanism of Mitochondrial Connexin43′s Protection of the Neurovascular Unit under Acute Cerebral Ischemia-Reperfusion Injury

    PubMed Central

    Hou, Shuai; Shen, Ping-Ping; Zhao, Ming-Ming; Liu, Xiu-Ping; Xie, Hong-Yan; Deng, Fang; Feng, Jia-Chun

    2016-01-01

    We observed mitochondrial connexin43 (mtCx43) expression under cerebral ischemia-reperfusion (I/R) injury, analyzed its regulation, and explored its protective mechanisms. Wistar rats were divided into groups based on injections received before middle cerebral artery occlusion (MCAO). Cerebral infarction volume was detected by 2,3,5-triphenyltetrazolim chloride staining, and cell apoptosis was observed by transferase dUTP nick end labeling. We used transmission electron microscopy to observe mitochondrial morphology and determined superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. MtCx43, p-mtCx43, protein kinase C (PKC), and p-PKC expression were detected by Western blot. Compared with those in the IR group, cerebral infarction volumes in the carbenoxolone (CBX) and diazoxide (DZX) groups were obviously smaller, and the apoptosis indices were down-regulated. Mitochondrial morphology was damaged after I/R, especially in the IR and 5-hydroxydecanoic acid (5-HD) groups. Similarly, decreased SOD activity and increased MDA were observed after MCAO; CBX, DZX, and phorbol-12-myristate-13-acetate (PMA) reduced mitochondrial functional injury. Expression of mtCx43 and p-mtCx43 and the p-Cx43/Cx43 ratio were significantly lower in the IR group than in the sham group. These abnormalities were ameliorated by CBX, DZX, and PMA. MtCx43 may protect the neurovascular unit from acute cerebral IR injury via PKC activation induced by mitoKATP channel agonists. PMID:27164087

  17. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43.

    PubMed

    Li, Nan; Mruk, Dolores D; Chen, Haiqi; Wong, Chris K C; Lee, Will M; Cheng, C Yan

    2016-01-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction. PMID:27436542

  18. Cardiac connexin-43 and PKC signaling in rats with altered thyroid status without and with omega-3 fatty acids intake.

    PubMed

    Szeiffová Bačová, B; Egan Beňová, T; Viczenczová, C; Soukup, T; Rauchová, H; Pavelka, S; Knezl, V; Barančík, M; Tribulová, N

    2016-09-19

    Thyroid hormones are powerful modulators of heart function and susceptibility to arrhythmias via both genomic and non-genomic actions. We aimed to explore expression of electrical coupling protein connexin-43 (Cx43) in the heart of rats with altered thyroid status and impact of omega-3 polyunsaturated fatty acids (omega-3) supplementation. Adult male Lewis rats were divided into following six groups: euthyroid controls, hyperthyroid (treated with T(3)) and hypothyroid (treated with methimazol) with or without six-weeks lasting supplementation with omega-3 (20 mg/100 g/day). Left and right ventricles, septum and atria were used for immunoblotting of Cx43 and protein kinase C (PKC). Total expression of Cx43 and its phosphorylated forms were significantly increased in all heart regions of hypothyroid rats compared to euthyroid controls. In contrast, the total levels of Cx43 and its functional phosphorylated forms were decreased in atria and left ventricle of hyperthyroid rats. In parallel, the expression of PKC epsilon that phosphorylates Cx43, at serine 368, was increased in hypothyroid but decreased in hyperthyroid rat hearts. Omega-3 intake did not significantly affect either Cx43 or PKC epsilon alterations. In conclusion, there is an inverse relationship between expression of cardiac Cx43 and the levels of circulating thyroid hormones. It appears that increased propensity of hyperthyroid while decreased of hypothyroid individuals to malignant arrhythmias may be in part attributed to the changes in myocardial Cx43. PMID:27643942

  19. Changes in phosphorylation of connexin43 in rats during acute myocardial hypoxia and effects of antiarrhythmic peptide on the phosphorylation.

    PubMed

    Wang, Rong; Zhang, Cuntai; Ruan, Yanfei; Liu, Nian; Wang, Lin

    2007-06-01

    In order to confirm the hypothesis that during acute hypoxia, the antiarrhythmic peptide (AAP10) could improve conductance by changing the phosphorylation state of connexin43 (Cx43), isolated perfused rat hearts were randomly divided into three groups: control, hypoxia and AAP10 (n=9 in each group). The change in Cx43 phosphorylation was tested by Western-blot; the distribution of Cx43 was observed by confocal immunofluorescence microscopy. Western-blot analysis revealed that the expression of total Cx43 protein was significantly decreased during acute hypoxia, while nonphosphorylated Cx43 (NP-Cx43) was unchanged. AAP10 could increase the expression of total Cx43 protein, but had no effects on the NP-Cx43 protein. Immunofluorescence study showed that during acute hypoxia, both total Cx43 and NP-Cx43 proteins were greatly decreased, while AAP10 only increased the expression of total Cx43 protein, but had no effect of the NP-Cx43 protein expression. These findings suggested that the decrease of intercellular communication may be associated with the reduction of phosphorylated Cx43 (p-Cx43) and translocation of NP-Cx43 from the surface of gap junction into intracellular pools during acute hypoxia. AAP10 can improve intercellular communication by enhancing phosphorylation of Cx43.

  20. Connexin43 Forms Supramolecular Complexes through Non-Overlapping Binding Sites for Drebrin, Tubulin, and ZO-1

    PubMed Central

    Ambrosi, Cinzia; Ren, Cynthia; Spagnol, Gaelle; Cavin, Gabriel; Cone, Angela; Grintsevich, Elena E.; Sorgen, Paul L.

    2016-01-01

    Gap junctions are membrane specialization domains identified in most tissue types where cells abut each other. The connexin channels found in these membrane domains are conduits for direct cell-to-cell transfer of ions and molecules. Connexin43 (Cx43) is the most ubiquitous connexin, with critical roles in heart, skin, and brain. Several studies described the interaction between Cx43 and the cytoskeleton involving the actin binding proteins Zonula occludens (ZO-1) and drebrin, as well as with tubulin. However, a direct interaction has not been identified between drebrin and Cx43. In this study, co-IP and NMR experiments were used to demonstrate that the Cx43-CT directly interacts with the highly conserved N-terminus region of drebrin. Three Cx43-CT areas were found to be involved in drebrin binding, with residues 264–275 being critical for the interaction. Mimicking Src phosphorylation within this region (Y265) significantly disrupted the interaction between the Cx43-CT and drebrin. Immunofluorescence showed colocalization of Cx43, drebrin, and F-actin in astrocytes and Vero cells membrane, indicating that Cx43 forms a submembrane protein complex with cytoskeletal and scaffolding proteins. The co-IP data suggest that Cx43 indirectly interacts with F-actin through drebrin. Along with the known interaction of the Cx43-CT with ZO-1 and tubulin, the data presented here for drebrin indicate non-overlapping and separated binding sites for all three proteins for which simultaneous binding could be important in regulating cytoskeleton rearrangements, especially for neuronal migration during brain development. PMID:27280719

  1. Beta Lactams Antibiotic Ceftriaxone Modulates Seizures, Oxidative Stress and Connexin 43 Expression in Hippocampus of Pentylenetetrazole Kindled Rats

    PubMed Central

    Hussein, Abdelaziz M.; Ghalwash, Mohammed; Magdy, Khaled; Abulseoud, Osama A.

    2016-01-01

    Background and Purpose: This study aimed to investigate the effect of ceftriaxone on oxidative stress and gap junction protein (connexin 43, Cx-43) expression in pentylenetetrazole (PTZ) induced kindling model. Methods: Twenty four Sprague dawely rats were divided into 3 equal groups (a) normal group: normal rats. (b) PTZ kindled group: received PTZ at the dose of 50 mg/kg via intraperitoneal injection (i.p.) every other day for 2 weeks (c) ceftriaxone treated group: received ceftriaxone at the dose 200 mg\\kg/12 hrs via i.p. injection daily from the 6th dose of PTZ for 3 days. Racine score, latency before beginning the first myoclonic jerk and duration of the jerks used as parameters of behavioral assessment. Immunohistopathological study for Cx-43 expression in hippocampus and measurement of markers of oxidative stress (malondialdehyde [MDA], low reduced glutathione [GSH] and catalase [CAT]) in hippocampal neurons were done. Results: PTZ kindling was associated with behavioral changes (in the form high stage of Racine score, long seizure duration and short latency for the first jerk), enhanced oxidative stress state (as demonstrated by high MDA, low GSH and CAT) and up regulation of Cx43 in hippocampal regions. While, ceftriaxone treatment ameliorated, significantly, PTZ-induced convulsions and caused significant improvement in oxidative stress markers and Cx-43 expression in hippocamal regions (p < 0.05). Conclusions: These findings support the anticonvulsive effects of some beta-lactams antibiotics which could offer a possible contributor in the basic treatment of temporal lobe epilepsy. This effect might be due to reduction of oxidative stress and Cx43 expression. PMID:27390674

  2. Expression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus).

    PubMed

    Li, Shuo; Peng, Weijiao; Chen, Xiaoli; Geng, Xuyun; Zhan, Wenbin; Sun, Jinsheng

    2016-08-01

    Connexin43 (Cx43) is the best characterized gap junction protein that allows the direct exchange of signaling molecules during cell-to-cell communications. The immunological functions and ATP permeable properties of Cx43 have been insensitively examined in mammals. The similar biological significance of Cx43 in lower vertebrates, however, is not yet understood. In the present study we identified and characterized a Cx43 ortholog (termed PoCx43) from Japanese flounder (Paralichthys olivaceus) and investigated its role in immune challenge-induced extracellular ATP release. PoCx43 mRNA transcripts are widely distributed in all tested normal tissues and cells with predominant expression in the brain, and are significantly up-regulated by LPS, poly(I:C) and zymosan challenges and Edwardsiella tarda infections as well, suggesting that PoCx43 expression was modulated by the inflammatory stresses. In addition, cyclic AMP (cAMP), an essential second messenger, also plays an important role in regulating PoCx43 gene expression, by which the PoCx43-mediated gap junctional communication may be regulated. Furthermore, overexpression of PoCx43 in Japanese flounder FG-9307 cells significantly potentiates the LPS- and poly(I:C)-induced extracellular ATP release and this enhanced ATP release was attenuated by pre-incubation with Cx43 inhibitor carbenoxolone. In a complementary experiment, down-regulation of PoCx43 endogenous expression in FG-9307 cells with small interfering RNA also significantly reduced the PAMP-induced extracellular ATP release, suggesting that PoCx43 is an important ATP release conduit under the immune challenge conditions. Finally, we showed that extracellular ATP stimulation led to an increased PoCx43 expression which probably provides a feedback mechanism in regulating PoCx43 expression at the transcriptional level. These findings suggest that PoCx43 is an inducible immune response gene and an important conduit for immune challenge-induced extracellular ATP

  3. Pregnancy enhances sustained Ca2+ bursts and endothelial nitric oxide synthase activation in ovine uterine artery endothelial cells through increased connexin 43 function.

    PubMed

    Yi, Fu-Xian; Boeldt, Derek S; Gifford, Shannon M; Sullivan, Jeremy A; Grummer, Mary A; Magness, Ronald R; Bird, Ian M

    2010-01-01

    Endothelium-mediated vasodilation is specifically enhanced in uterine circulation during pregnancy, and production of nitric oxide (NO) is increased in response to a wide array of agonists. Uterine artery endothelial cells from nonpregnant (NP-UAECs) or pregnant (P-UAECs) ewes maintained in culture still show a pregnancy-enhanced difference in ATP-stimulated endothelial NO synthase (eNOS; official symbol NOS3) activation, even though NOS3 protein, purinergic receptors, and associated cell signaling proteins are expressed at equal levels. We have also shown that the pregnancy-enhanced endothelial cell NO response to ATP requires an enhanced and sustained capacitative entry phase that is likely mediated via canonical transient receptor potential protein/inositol 1,4,5-trisphosphate receptor type 2 interaction. In this study, we now show by simultaneous video imaging of individual Fura-2-loaded cells that the pregnancy-enhanced capacitative entry phase is not continuous and equal in all cells, but is in fact mediated as a series of periodic [Ca(2+)](i) bursts within individual cells. Not only does pregnancy increase the number of bursts over a longer time period in individual cells, but also a greater proportion of cells exhibit this burst activity, and at high cell density this occurs in a synchronous manner. The mediator of cell synchronization is connexin 43 (Cx43) gap junctions because 1) Cx43 is readily detectable by Western blot analysis in UAECs, whereas Cx40 and Cx37 are weakly detected or absent, and 2) pregnancy-specific enhancement of [Ca(2+)](i) bursts by ATP is blocked by inhibitory loop peptides selective to Cx43 ((43,37)GAP27) but not by a scrambled control peptide or (40)GAP27 or (40,37)GAP26 peptides, which are specific to Cx40 or Cx37. The relationship between Ca(2+) bursts and NOS3 activation is further established by the finding that (43,37)GAP27 inhibits ATP-stimulated NOS3 activation but has no effect on cell mitogenesis. We conclude that it is

  4. Expression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus).

    PubMed

    Li, Shuo; Peng, Weijiao; Chen, Xiaoli; Geng, Xuyun; Zhan, Wenbin; Sun, Jinsheng

    2016-08-01

    Connexin43 (Cx43) is the best characterized gap junction protein that allows the direct exchange of signaling molecules during cell-to-cell communications. The immunological functions and ATP permeable properties of Cx43 have been insensitively examined in mammals. The similar biological significance of Cx43 in lower vertebrates, however, is not yet understood. In the present study we identified and characterized a Cx43 ortholog (termed PoCx43) from Japanese flounder (Paralichthys olivaceus) and investigated its role in immune challenge-induced extracellular ATP release. PoCx43 mRNA transcripts are widely distributed in all tested normal tissues and cells with predominant expression in the brain, and are significantly up-regulated by LPS, poly(I:C) and zymosan challenges and Edwardsiella tarda infections as well, suggesting that PoCx43 expression was modulated by the inflammatory stresses. In addition, cyclic AMP (cAMP), an essential second messenger, also plays an important role in regulating PoCx43 gene expression, by which the PoCx43-mediated gap junctional communication may be regulated. Furthermore, overexpression of PoCx43 in Japanese flounder FG-9307 cells significantly potentiates the LPS- and poly(I:C)-induced extracellular ATP release and this enhanced ATP release was attenuated by pre-incubation with Cx43 inhibitor carbenoxolone. In a complementary experiment, down-regulation of PoCx43 endogenous expression in FG-9307 cells with small interfering RNA also significantly reduced the PAMP-induced extracellular ATP release, suggesting that PoCx43 is an important ATP release conduit under the immune challenge conditions. Finally, we showed that extracellular ATP stimulation led to an increased PoCx43 expression which probably provides a feedback mechanism in regulating PoCx43 expression at the transcriptional level. These findings suggest that PoCx43 is an inducible immune response gene and an important conduit for immune challenge-induced extracellular ATP

  5. Gliotoxin potentiates osteoblast differentiation by inhibiting nuclear factor-κB signaling

    PubMed Central

    WANG, GUANGYE; ZHANG, XIAOHAI; YU, BAOQING; REN, KE

    2015-01-01

    The differentiation of pluripotent mesenchymal stem cells to mature osteoblasts is crucial for the maintenance of the adult skeleton. In rheumatic arthritis, osteoblast differentiation is impaired by the overproduction of cytokine tumor necrosis factor (TNF)-α. It has been demonstrated that TNF-α is able to inhibit osteoblast differentiation through the activation of nuclear factor (NF)-κB signaling. As a result of the critical role of TNF-α and NF-κB in the pathogenesis of bone-loss associated diseases, these factors are regarded as key targets for the development of therapeutic agents. In the current study, the role of the NF-κB inhibitor gliotoxin (GTX) in the regulation of osteoblast differentiation was evaluated. The non-toxic GTX doses were determined to be ≤3 μg/ml. It was revealed that GTX was able to block TNF-α-induced inhibition of osteoblast differentiation, as indicated by alkaline phosphatase (ALP) activity and ALP staining assays, as well as the expression levels of osteoblast-associated genes Col I, Ocn, Bsp, Runx2, Osx and ATF4. Additionally, it was identified that gliotoxin directly promoted bone morphoge-netic protein-2-induced osteoblast differentiation. GTX was found to inhibit the accumulation of NF-κB protein p65 in the nucleus and reduce NF-κB transcriptional activity, suggesting that GTX potentiated osteoblast differentiation via the suppression of NF-κB signaling. PMID:25816130

  6. Inhibition of Connexin 43 Hemichannel-Mediated ATP Release Attenuates Early Inflammation During the Foreign Body Response

    PubMed Central

    Calder, Bennett W.; Rhett, Joshua Matthew; Bainbridge, Heather; Fann, Stephen A.; Gourdie, Robert G.

    2015-01-01

    Background: In the last 50 years, the use of medical implants has increased dramatically. Failure of implanted devices and biomaterials is a significant source of morbidity and increasing healthcare expenditures. An important cause of implant failure is the host inflammatory response. Recent evidence implicates extracellular ATP as an important inflammatory signaling molecule. A major pathway for release of cytoplasmic ATP into the extracellular space is through connexin hemichannels, which are the unpaired constituents of gap junction intercellular channels. Blockade of hemichannels of the connexin 43 (Cx43) isoform has been shown to reduce inflammation and improve healing. We have developed a Cx43 mimetic peptide (JM2) that targets the microtubule-binding domain of Cx43. The following report investigates the role of the Cx43 microtubule-binding domain in extracellular ATP release by Cx43 hemichannels and how this impacts early inflammatory events of the foreign body reaction. Methods: In vitro Cx43 hemichannel-mediated ATP release by cultured human microvascular endothelial cells subjected to hypocalcemic and normocalcemic conditions was measured after application of JM2 and the known hemichannel blocker, flufenamic acid. A submuscular silicone implant model was used to investigate in vivo ATP signaling during the early foreign body response. Implants were coated with control pluronic vehicle or pluronic carrying JM2, ATP, JM2+ATP, or known hemichannel blockers and harvested at 24 h for analysis. Results: JM2 significantly inhibited connexin hemichannel-mediated ATP release from cultured endothelial cells. Importantly, the early inflammatory response to submuscular silicone implants was inhibited by JM2. The reduction in inflammation by JM2 was reversed by the addition of exogenous ATP to the pluronic vehicle. Conclusions: These data indicate that ATP released through Cx43 hemichannels into the vasculature is an important signal driving the early inflammatory

  7. MicroRNA-1 downregulation increases connexin 43 displacement and induces ventricular tachyarrhythmias in rodent hypertrophic hearts.

    PubMed

    Curcio, Antonio; Torella, Daniele; Iaconetti, Claudio; Pasceri, Eugenia; Sabatino, Jolanda; Sorrentino, Sabato; Giampà, Salvatore; Micieli, Mariella; Polimeni, Alberto; Henning, Beverley J; Leone, Angelo; Catalucci, Daniele; Ellison, Georgina M; Condorelli, Gianluigi; Indolfi, Ciro

    2013-01-01

    Downregulation of the muscle-specific microRNA-1 (miR-1) mediates the induction of pathologic cardiac hypertrophy. Dysfunction of the gap junction protein connexin 43 (Cx43), an established miR-1 target, during cardiac hypertrophy leads to ventricular tachyarrhythmias (VT). However, it is still unknown whether miR-1 and Cx43 are interconnected in the pro-arrhythmic context of hypertrophy. Thus, in this study we investigated whether a reduction in the extent of cardiac hypertrophy could limit the pathological electrical remodeling of Cx43 and the onset of VT by modulating miR-1 levels. Wistar male rats underwent mechanical constriction of the ascending aorta to induce pathologic left ventricular hypertrophy (LVH) and afterwards were randomly assigned to receive 10mg/kg valsartan, VAL (LVH+VAL) delivered in the drinking water or placebo (LVH) for 12 weeks. Sham surgery was performed for control groups. Programmed ventricular stimulation reproducibly induced VT in LVH compared to LVH+VAL group. When compared to sham controls, rats from LVH group showed a significant decrease of miR-1 and an increase of Cx43 expression and its ERK1/2-dependent phosphorylation, which displaces Cx43 from the gap junction. Interestingly, VAL administration to rats with aortic banding significantly reduced cardiac hypertrophy and prevented miR-1 down-regulation and Cx43 up-regulation and phosphorylation. Gain- and loss-of-function experiments in neonatal cardiomyocytes (NCMs) in vitro confirmed that Cx43 is a direct target of miR-1. Accordingly, in vitro angiotensin II stimulation reduced miR-1 levels and increased Cx43 expression and phosphorylation compared to un-stimulated NCMs. Finally, in vivo miR-1 cardiac overexpression by an adenoviral vector intra-myocardial injection reduced Cx43 expression and phosphorylation in mice with isoproterenol-induced LVH. In conclusion, miR-1 regulates Cx43 expression and activity in hypertrophic cardiomyocytes in vitro and in vivo. Treatment of

  8. Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo.

    PubMed

    Wrenzycki, C; Herrmann, D; Carnwath, J W; Niemann, H

    1996-09-01

    In this study we have examined the presence of mRNA encoding connexin 43 (Cx43) in bovine embryos derived in vivo and in vitro. Cumulus-oocyte complexes, immature and matured oocytes liberated from cumulus cells, zygotes, 2-4-cell and 8-16-cell embryos, morulae, blastocysts and hatched blastocysts were produced in vitro from ovaries obtained from an abattoir using TCM 199 supplemented with hormones and 10% oestrous cow serum for maturation. Cumulus-oocyte complexes matured for 24 h were exposed to bull spermatozoa for 19 h and then cultured in TCM 199 supplemented with 10% oestrous cow serum to the desired developmental stage. Morulae and blastocysts derived in vivo were collected from superovulated donor cows. Total RNA was extracted from pools of 60-200 bovine oocytes or embryos using a modified phenol-chloroform extraction method and analysed by reverse transcriptase polymerase chain reaction. Before reverse transcription, aliquots of DNase-digested embryonic RNA were tested by polymerase chain reaction using bovine-specific primers to control for residual genomic DNA contamination. DNA-free, total RNA was reverse transcribed after preincubation with the Cx43 specific 3'primer. The resultant cDNA was amplified by polymerase chain reaction using Cx43 specific primers that define a 516 bp fragment of Cx43. The reverse transcriptase polymerase chain reaction product was verified by restriction enzyme analysis with Alu I and sequencing. Assays were repeated at least twice for each developmental stage and provided identical results between replicates. Cx43 transcripts were detected in bovine morulae and blastocysts grown in vivo. In contrast, whereas the early in vitro stages from cumulus-oocyte complexes to morulae expressed Cx43, blastocysts and hatched blastocysts did not have detectable concentrations of mRNA from this gene. Restriction enzyme cutting revealed three fragments of the predicted size (139, 177, 200 bp). The amplified product showed 100% identity

  9. Changes in integrin αv, vinculin and connexin43 in the medial prefrontal cortex in rats under single-prolonged stress.

    PubMed

    Li, Yana; Han, Fang; Shi, Yuxiu

    2015-04-01

    Post‑traumatic stress disorder (PTSD) is a stress‑accociated mental disorder that occurs as a result of exposure to a traumatic event, with characteristic symptoms, including intrusive memories, hyperarousal and avoidance. The medial prefrontal cortex (mPFC) is known to be significantly involved in emotional adjustment, particularly introspection, inhibition of the amygdala and emotional memory. Previous structural neuroimaging studies have revealed that the mPFC of PTSD patients was significantly smaller when compared with that of controls and their emotional adjustment function was weakened. However, the mechanisms that cause such atrophy remain to be elucidated. The aim of the present study was to elucidate the possible mechanisms involved in apoptosis induced by single‑prolonged stress (SPS) in the mPFC of PTSD rats. SPS is an animal model reflective of PTSD. Of the proposed animal models of PTSD, SPS is one that has been shown to be reliably reproducible in patients with PTSD. Wistar rats were sacrificed at 1, 4, 7 and 14 days after exposure to SPS. Apoptotic cells were assessed using electron microscopy and the TUNEL method. Expression of integrin αv, vinculin and connexin43 were detected using immunohistochemistry, western blotting and reverse transcription polymerase chain reaction. The present results demonstrated that apoptotic cells significantly increased in the mPFC of SPS rats, accompanied with changes in expression of integrin αv, vinculin and connexin43. The present results indicated that SPS‑induced apoptosis in the mPFC of PTSD rats and the mitochondrial pathway were involved in the process of SPS‑induced apoptosis.

  10. Quantitative Immunohistochemistry of Desmosomal Proteins (Plakoglobin, Desmoplakin and Plakophilin), Connexin-43, and N-cadherin in Arrhythmogenic Cardiomyopathy: An Autopsy Study

    PubMed Central

    Tavora, Fabio; Zhang, Mingchang; Cresswell, Nathaniel; Li, Ling; Fowler, David; Franco, Marcello; Burke, Allen

    2013-01-01

    Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder related to mutations in desmosomal proteins. The current study tests the hypothesis that immunohistochemical staining for desmosomal proteins is of diagnostic utility by studying autopsy-confirmed cases of ARVC. Methods and Results: We studied 23 hearts from patients dying suddenly with ARVC. Control subject tissues were 21 hearts from people dying from non-cardiac causes (n=15), dilated cardiomyopathy (n=3) and coronary artery disease (n=3). Areas free of fibrofatty change or scarring were assessed on 50 sections from ARVC (24 left ventricle, 26 right ventricle) and 28 sections from controls. Immunohistochemical stains against plakoglobin, plakophilin, desmoplakin, connexin-43, and N-cadherin were applied and area expression analyzed by computerized morphometry. Desmin was stained as a control for fixation and similarly analyzed. The mean area of desmin expression was similar in controls and ARVC (86% vs. 85%, p=0.6). Plakoglobin expression was 4.9% ± 0.3% in controls, vs. 4.6% ± 0.3% in ARVC (p=0.3). Plakophilin staining was 4.8% ± 0.3% in controls vs. 4.4% ± 03% in ARVC (p=0.3). Desmoplakin staining was 3.4% in controls vs. 3.2 ± 0.2% in ARVC (p=0.6). There were no significant differences when staining was compared between right and left ventricles (all p > 0.1). For non-desmosomal proteins, the mean area of connexin-43 staining showed no significant difference by presence of disease. Conclusions: The small and insignificant decrease in junction protein expression in ARVC suggests that immunohistochemistry is not a useful tool for the diagnosis. PMID:23802019

  11. Gap junctional communication modulates gene transcription by altering the recruitment of Sp1 and Sp3 to connexin-response elements in osteoblast promoters

    NASA Technical Reports Server (NTRS)

    Stains, Joseph P.; Lecanda, Fernando; Screen, Joanne; Towler, Dwight A.; Civitelli, Roberto

    2003-01-01

    Loss-of-function mutations of gap junction proteins, connexins, represent a mechanism of disease in a variety of tissues. We have shown that recessive (gene deletion) or dominant (connexin45 overexpression) disruption of connexin43 function results in osteoblast dysfunction and abnormal expression of osteoblast genes, including down-regulation of osteocalcin transcription. To elucidate the molecular mechanisms of gap junction-sensitive transcriptional regulation, we systematically analyzed the rat osteocalcin promoter for sensitivity to gap junctional intercellular communication. We identified an Sp1/Sp3 containing complex that assembles on a minimal element in the -70 to -57 region of the osteocalcin promoter in a gap junction-dependent manner. This CT-rich connexin-response element is necessary and sufficient to confer gap junction sensitivity to the osteocalcin proximal promoter. Repression of osteocalcin transcription occurs as a result of displacement of the stimulatory Sp1 by the inhibitory Sp3 on the promoter when gap junctional communication is perturbed. Modulation of Sp1/Sp3 recruitment also occurs on the collagen Ialpha1 promoter and translates into gap junction-sensitive transcriptional control of collagen Ialpha1 gene expression. Thus, regulation of Sp1/Sp3 recruitment to the promoter may represent a potential general mechanism for transcriptional control of target genes by signals passing through gap junctions.

  12. Influence of the antiandrogen flutamide on connexin 43 (Cx43) gene and protein expression in the porcine placenta and uterus during pregnancy.

    PubMed

    Wieciech, Iwona; Grzesiak, Małgorzata; Knapczyk-Stwora, Katarzyna; Pytlik, Anna; Słomczynska, Maria

    2014-01-01

    The study focuses on the expression of connexin 43 (Cx43), a gap junctional protein in the porcine placenta and uterus. The aim was to examine Cx43 mRNA and protein expression after antiandrogen flutamide treatment. Flutamide was injected into pregnant gilts at a daily dose of 50 mg/kg body weight at different stages of pregnancy: between days 43-49 (50 dpc), 83-89 (90 dpc) and 101-107 (108 dpc) of gestation. The animals were sacrificed and tissues were collected one day after the last injection. Cx43 immunostaining was observed in epithelial and stromal cells of the fetal part of the placenta; luminal and glandular epithelial cells of maternal part of the placenta and myometrium of the uterus within placentation sites. Cx43 was also found in glandular epithelium and myometrium of non-placental uterus. Flutamide treatment caused fluctuations in Cx43 expression especially before parturition. Although significant changes in Cx43 mRNA expression were observed only in the fetal part of the placenta, Cx43 protein level was affected within the maternal part of the placenta and non-placental uterus. These results suggest the involvement of androgens in the regulation of Cx43 expression within the feto-maternal compartment in pigs. However, androgen deficiency caused pronounced changes during late pregnancy and before parturition. These results are interesting due to the functional changes in the porcine uterus during the preparturient period that is determined by Cx43 protein.

  13. Ageing related down-regulation of myocardial connexin-43 and up-regulation of MMP-2 may predict propensity to atrial fibrillation in experimental animals.

    PubMed

    Nagibin, V; Egan Benova, T; Viczenczova, C; Szeiffova Bacova, B; Dovinova, I; Barancik, M; Tribulova, N

    2016-09-19

    Mechanisms underlying atrial fibrillation (AF), the most common cardiac arrhythmia, particularly in aged population, are not fully elucidated. We have previously shown an increased propensity of old guinea pigs (GPs) heart to inducible AF when comparing to young animals. This study aimed to verify our hypothesis that susceptibility of aged heart to AF may be attributed to abnormalities in myocardial connexin-43 (Cx43) and extracellular matrix that affect cardiac electrical properties. Experiments were conducted on male and female 4-week-old and 24-week-old GPs. Atrial tissue was processed for analysis of Cx43 topology using immunohistochemistry, expression of Cx43 protein using immunobloting, and expression of mRNA of Cx43 and extracellular matrix metalloproteinase-2 (MMP-2) using real time PCR. Immunohistochemistry revealed uniform Cx43 distribution predominantly on lateral sides of the cardiomyocytes of young male and female GP atria. In contrast, non-uniform distribution, mislocalization and reduced immunolabeling of Cx43 were detected in atria of old GPs. In parallel, the atrial tissue levels of Cx43 mRNA were significantly decreased, while mRNA expression of MMP-2 was significantly increased in old versus young GPs. The changes were more pronounced in old GPs males comparing to females. Findings indicate that age-related down-regulation of atrial Cx43 and up-regulation of MMP-2 as well as disordered Cx43 distribution can facilitate development of AF in old guinea pig hearts. PMID:27643943

  14. Anti-arrhythmic effects of atrial ganglionated plexi stimulation is accompanied by preservation of connexin43 protein in ischemia-reperfusion canine model

    PubMed Central

    Wang, Songyun; Li, Hewei; Yu, Lilei; Chen, Mingxian; Wang, Zhuo; Huang, Bing; Zhou, Liping; Zhou, Xiaoya; Jiang, Hong

    2015-01-01

    Background: Vagal nerve stimulation (VNS) has been shown to provide a protective effect against ischemia/reperfusion (I/R)-related arrhythmias by preventing the loss of Connexin43 (Cx43). Our previous studies showed that atrial epicardial ganglionated plexus stimulation (GPS) might exert a VNS-like effect on ventricular electrophysiology. Objectives: To investigate whether GPS could preserve Cx43 and reduce I/R induced ventricular arrhythmia. Methods: Sixteen dogs were randomly divided into GPS group (N = 8, receiving GPS) and Sham group (N = 8, receiving sham GPS). Ventricular effective refractory period (ERP) and heart rate variability (HRV) were measured at baseline and 1 h after GPS. Myocardial I/R was then performed. Ventricular arrhythmia occurred during the first hour after reperfusion was measured and myocardial tissue from the peri-infarct zone was excised for immunohistological analysis. In another 4 dogs (Control group, receiving sham GPS and sham I/R), myocardial tissue from the corresponding area was also excised. Results: Compared with the Sham group, GPS caused a significant increase in ventricular ERP and HRV, and a significant decrease in I/R-induced ventricular arrhythmias. Western blotting revealed a marked reduction in the amount of phosphorylated Cx43 and total Cx43 in the Sham group, whereas no significant change was observed in the GPS group compared with the Control group. Immunohistochemistry results confirmed that the myocardial I/R-induced loss of phosphorylated Cx43 from the intercellular junctions was prevented by GPS. Conclusion: GPS protects against I/R induced ventricular arrhythmias, accompanied by preserving Cx43. PMID:26885184

  15. Mechanism of PKA-dependent and lipid-raft independent stimulation of Connexin43 expression by oxytoxin in mouse embryonic stem cells.

    PubMed

    Yun, Seung Pil; Park, Su Shin; Ryu, Jung Min; Park, Jae Hong; Kim, Mi Ok; Lee, Jang-Hern; Han, Ho Jae

    2012-07-01

    Previous studies shows that connexins appear very early during murine embryo development, the gap junctional intercellular communication found in the inner cell mass of early embryo is also maintained in embryonic stem cells (ESC), and expression of oxytocin receptor (OTR) is developmentally regulated at early embryonic development. However, effect of oxytocin (OT) on the regulation of the connexin43 (Cx43) and maintenance of undifferentiation is not fully understood in stem cells. Therefore, we investigated the effect of OT on Cx43 expression and related signaling cascades in mouse ESC. OT increased Cx43 expression that was inhibited by the OTR inhibitor atosiban. In experiments to examine whether the effect of OT depends on lipid rafts, caveolin-1 (cav-1), cav-2, and flotillin-2, but not OTR, were detected in lipid raft fractions. Also, colocalization of OTR, cav-1, and cav-2 was not detected. Moreover, the lipid raft disruptor methyl-β-cyclodextrin did not attenuate OT-induced Cx43 expression. In experiments to examine related signaling pathways, OT activated cAMP/protein kinase A (PKA) which was inhibited by adenylyl cyclase inhibitor SQ 22536 and PKA inhibitor PKI. OT increased nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) phosphorylation which was inhibited by PKI. OT also increased cAMP response element-binding (CREB)/CREB-binding protein (CBP) expression in the nucleus and induced the formation of CREB1/NF-κB/CBP complexes, which was blocked by the NF-κB-specific small interfering RNA, NF-κB inhibitors, SN50, and bay11-7082. Complex disruption by NF-κB inhibitors decreased OT-induced Cx43 expression. In conclusion, OT stimulates Cx43 expression through the NF-κB/CREB/CBP complex via the lipid raft-independent OTR-mediated cAMP/PKA in mouse ESC.

  16. Prognostic Impact of Reduced Connexin43 Expression and Gap Junction Coupling of Neoplastic Stromal Cells in Giant Cell Tumor of Bone

    PubMed Central

    Balla, Peter; Maros, Mate Elod; Barna, Gabor; Antal, Imre; Papp, Gergo; Sapi, Zoltan; Athanasou, Nicholas Anthony; Benassi, Maria Serena; Picci, Pierro; Krenacs, Tibor

    2015-01-01

    Missense mutations of the GJA1 gene encoding the gap junction channel protein connexin43 (Cx43) cause bone malformations resulting in oculodentodigital dysplasia (ODDD), while GJA1 null and ODDD mutant mice develop osteopenia. In this study we investigated Cx43 expression and channel functions in giant cell tumor of bone (GCTB), a locally aggressive osteolytic lesion with uncertain progression. Cx43 protein levels assessed by immunohistochemistry were correlated with GCTB cell types, clinico-radiological stages and progression free survival in tissue microarrays of 89 primary and 34 recurrent GCTB cases. Cx43 expression, phosphorylation, subcellular distribution and gap junction coupling was also investigated and compared between cultured neoplastic GCTB stromal cells and bone marow stromal cells or HDFa fibroblasts as a control. In GCTB tissues, most Cx43 was produced by CD163 negative neoplastic stromal cells and less by CD163 positive reactive monocytes/macrophages or by giant cells. Significantly less Cx43 was detected in α-smooth muscle actin positive than α-smooth muscle actin negative stromal cells and in osteoclast-rich tumor nests than in the adjacent reactive stroma. Progressively reduced Cx43 production in GCTB was significantly linked to advanced clinico-radiological stages and worse progression free survival. In neoplastic GCTB stromal cell cultures most Cx43 protein was localized in the paranuclear-Golgi region, while it was concentrated in the cell membranes both in bone marrow stromal cells and HDFa fibroblasts. In Western blots, alkaline phosphatase sensitive bands, linked to serine residues (Ser369, Ser372 or Ser373) detected in control cells, were missing in GCTB stromal cells. Defective cell membrane localization of Cx43 channels was in line with the significantly reduced transfer of the 622 Da fluorescing calcein dye between GCTB stromal cells. Our results show that significant downregulation of Cx43 expression and gap junction coupling in

  17. RhoA/rho kinase signaling reduces connexin43 expression in high glucose-treated glomerular mesangial cells with zonula occludens-1 involvement

    SciTech Connect

    Xie, Xi; Chen, Cheng; Huang, Kaipeng; Wang, Shaogui; Hao, Jie; Huang, Junying; Huang, Heqing

    2014-10-01

    RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose-treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and an enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose-treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs. - Highlights: • RhoA/ROCK signaling induces Cx43 degradation in GMCs. • F-actin and ZO-1 have functions in the regulation of Cx43 by RhoA/ROCK signaling. • We reveal the relationship between RhoA/ROCK and Cx43 in the activation of NF-κB.

  18. Common genetic variation near the connexin-43 gene is associated with resting heart rate in African Americans: A genome-wide association study of 13,372 participants

    PubMed Central

    Deo, R.; Nalls, M.A.; Avery, C.L.; Smith, J.G.; Evans, D.S.; Keller, M.F.; Butler, A.M.; Buxbaum, S.G.; Li, G.; Quibrera, P. Miguel; Smith, E.N.; Tanaka, T.; Akylbekova, E.L.; Alonso, A.; Arking, D.E.; Benjamin, E.J.; Berenson, G.S.; Bis, J.C.; Chen, L.Y.; Chen, W.; Cummings, S.R.; Ellinor, P.T.; Evans, M.K.; Ferrucci, L.; Fox, E.R.; Heckbert, S.R.; Heiss, G.; Hsueh, W.C.; Kerr, K.F.; Limacher, M.C.; Liu, Y.; Lubitz, S.A.; Magnani, J.W.; Mehra, R.; Marcus, G.M.; Murray, S.S.; Newman, A.B.; Njajou, O.; North, K.E.; Paltoo, D.N.; Psaty, B.M.; Redline, S.S.; Reiner, A.P.; Robinson, J.G.; Rotter, J.I.; Samdarshi, T.E.; Schnabel, R.B.; Schork, N.J.; Singleton, A.B.; Siscovick, D.; Soliman, E.Z.; Sotoodehnia, N.; Srinivasan, S.R.; Taylor, H.A.; Trevisan, M.; Zhang, Z.; Zonderman, A.B.; Newton-Cheh, C.; Whitsel, E.A.

    2013-01-01

    BACKGROUND Genome-wide association studies have identified several genetic loci associated with variation in resting heart rate in European and Asian populations. No study has evaluated genetic variants associated with heart rate in African Americans. OBJECTIVE To identify novel genetic variants associated with resting heart rate in African Americans. METHODS Ten cohort studies participating in the Candidate-gene Association Resource and Continental Origins and Genetic Epidemiology Network consortia performed genome-wide genotyping of single nucleotide polymorphisms (SNPs) and imputed 2,954,965 SNPs using HapMap YRI and CEU panels in 13,372 participants of African ancestry. Each study measured the RR interval (ms) from 10-second resting 12-lead electrocardiograms and estimated RR-SNP associations using covariate-adjusted linear regression. Random-effects meta-analysis was used to combine cohort-specific measures of association and identify genome-wide significant loci (P ≤ 2.5 × 10−8). RESULTS Fourteen SNPs on chromosome 6q22 exceeded the genome-wide significance threshold. The most significant association was for rs9320841 (+13 ms per minor allele; P = 4.98 × 10−15). This SNP was approximately 350 kb downstream of GJA1, a locus previously identified as harboring SNPs associated with heart rate in Europeans. Adjustment for rs9320841 also attenuated the association between the remaining 13 SNPs in this region and heart rate. In addition, SNPs in MYH6, which have been identified in European genome-wide association study, were associated with similar changes in the resting heart rate as this population of African Americans. CONCLUSIONS An intergenic region downstream of GJA1 (the gene encoding connexin 43, the major protein of the human myocardial gap junction) and an intragenic region within MYH6 are associated with variation in resting heart rate in African Americans as well as in populations of European and Asian origin. PMID:23183192

  19. Cellular uptake of lead in the blood-cerebrospinal fluid barrier: Novel roles of Connexin 43 hemichannel and its down-regulations via Erk phosphorylation.

    PubMed

    Song, Han; Zheng, Gang; Liu, Yang; Shen, Xue-Feng; Zhao, Zai-Hua; Aschner, Michael; Luo, Wen-Jing; Chen, Jing-Yuan

    2016-04-15

    As the structural basis of blood-cerebrospinal fluid barrier (BCB), epithelial cells in the choroid plexus (CP) are targets for lead (Pb). Pb is known to accumulate in the CP; however, the mechanism of Pb uptake in the choroidal epithelial cells remains unknown. Recently, hemichannels of Connexin 43 (Cx43), the most ubiquitously expressed gap junction proteins in the CP, were found to be important pathways for many substances. This study was designed to investigate the roles of Cx43 in Pb uptake in the epithelial cells. Autometallography was used to outline Pb's subcellular location, and the characteristics of Pb transport into CP cells, including concentration- and time-dependence were analyzed by atomic absorption spectroscopy. Knockdown/overexpression of Cx43 with transient siRNA/plasmids transfections before Pb exposure diminished/increased the Pb accumulation. In the Z310 cell-based doxycycline-inducible Cx43 expression cell line (iZCx43), doxycycline induced a significant increase (3-fold) in Pb uptake, corresponding to the increased Cx43 levels. Activation of Cx43 hemichannels by reduced serum conditions caused an increase of Pb concentrations. Cx43-induced Pb uptake was attenuated after blockage of Cx43 hemichannels with its inhibitor, carbenoxolone. Additionally, down-regulation of Cx43 protein levels by Pb exposure paralleled cellular Pb concentrations in the time study. Concomitantly, expressions of phosphor-Src and phosphor-Erk were both significantly increased by Pb. However, inactivation of Erk, not Src pathway, reversed Pb-induced downregulation of Cx43. Taken together, these data establish that Pb can accumulate in the BCB and validate the role of Cx43 hemichannel in Pb uptake and its regulations through Erk phosphorylation.

  20. Are Synchronized Changes in Connexin-43 and Caveolin-3 a Bystander Effect in a Phoneutria nigriventer Venom Model of Blood-Brain Barrier Breakdown?

    PubMed

    Soares, Edilene Siqueira; Mendonça, Monique Culturato Padilha; Rocha, Thalita; Kalapothakis, Evanguedes; da Cruz-Höfling, Maria Alice

    2016-08-01

    Upregulation of caveolin-3 (Cav-3) or connexin-43 (Cx43) in astrocytes has been associated with important brain pathologies. We used Phoneutria nigriventer spider venom (PNV), which induces blood-brain barrier breakdown in rats, in order to investigate Cav-3 and Cx43 expression in the cerebellum over critical periods of rat envenomation. By immunofluorescence, western blotting (WB), and transmission electron microscopy (TEM), we assessed changes at 1, 2, 5, 24, and 72 h post-venom. WB showed immediate increases in Cav-3 and Cx43 at 1 h (interval of greatest manifestations of envenomation) that persisted at 5 h (when there were signs of recovery) and peaked at 24 h when no signs of envenomation were detectable. At 2 and 72 h, Cav-3 was downregulated and Cx43 had returned to baseline. PNV markedly intensified Cx43 in molecular, Purkinje and granular layers and Cav-3 in astrocytes whose colocalization to increased GFAP suggests interaction between reactive astrogliosis and Cav-3 upregulation. TEM showed swollen perivascular astrocytic end-feet and synaptic contact alterations that had generally resolved by 72 h. It is uncertain whether such PNV-induced synchronized changes are an interactive effect between Cav-3 and Cx43, or a bystander effect. Evidences indicate that Cav-3 downregulation coupled to Cx43 return to baseline at 72 h when no signs of envenomation were visible, suggesting homeostasis reestablishment. This experimental model is relevant to studying mechanisms involved in neurological disorders associated with Cav-3 overexpression. PMID:27067308

  1. Novel Pharmacophores of Connexin43 Based on the “RXP” Series of Cx43-binding Peptides

    PubMed Central

    Verma, Vandana; Larsen, Bjarne Due; Coombs, Wanda; Lin, Xianming; Spagnol, Gaelle; Sorgen, Paul L; Taffet, Steven M; Delmar, Mario

    2009-01-01

    Gap junction pharmacology is a nascent field. Previous studies have identified molecules that enhance intercellular communication, and may offer potential for innovative antiarrhythmic therapy. However, their specific molecular target(s) and mechanism(s) of action remain unknown. Previously, we identified a 34-amino acid peptide (RXP-E) that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and action potential propagation block. These results supported the feasibility of a peptide-based pharmacology to Cx43, but the structure of the core active element in RXP-E, an essential step for pharmacological development, remained undefined. Here, we used a combination of molecular modeling, surface plasmon resonance, nuclear magnetic resonance and patch clamp strategies to define, for the first time, a unique ensemble of pharmacophores that bind Cx43CT and prevent closure of Cx43 channels. Two particular molecules are best representatives of this family: a cyclized heptapeptide (called CyRP-71), and a linear octapeptide of sequence RRNYRRNY. These two small compounds offer the first structural platform for the design of Cx43-interacting gap junction openers. Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap junction channel function. PMID:19556520

  2. Surface potential and osteoblast attraction to calcium phosphate compounds is affected by selected alkaline hydrolysis processing.

    PubMed

    Smith, I O; Baumann, M J; Obadia, L; Bouler, J-M

    2004-08-01

    This study examines the link(s) between the suspension behavior of calcium deficient apatites (CDAs) and biphasic calcium phosphate (BCP), as measured by the zeta-potential, with respect to both whole bone and osteoblasts. CDA is fabricated by hydrolyzing an acidic CaP such as dicalcium diphosphate dihydrate (DCPD; CaHPO4.2H2O) and has a structure and composition close to bone apatite. Sintering CDA results in the formation of BCP ceramics consisting of mixtures of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), with the HA/beta-TCP weight ratio proportional to the Ca/P ratio of CDA. The choice of the base for the DCPD hydrolysis allows various ionic partial substitution of the formed CDA. Na for Ca partial substitution is of interest because of the resulting improvement in mechanical properties of the resulting BCP ceramics and NH4OH was used as a negative control. The zeta-potential was measured for these materials and the stability of the ceramic to bone interaction calculated. zeta-potential values decrease for CDA(NH4OH) versus CDA(NaOH) and increase for BCP(NH4OH) versus BCP(NaOH). While results of these analyses indicate that NH4OH and NaOH processed CDA and BCP will likely yield osteoblast attachment in vivo, differences in the zeta-potentials may explain varying degrees of cell attachment.

  3. Gap junction endocytosis and lysosomal degradation of connexin43-P2 in WB-F344 rat liver epithelial cells treated with DDT and lindane.

    PubMed

    Guan, X; Ruch, R J

    1996-09-01

    Treatment of WB-F344 rat liver epithelial cells with DDT (1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane) or lindane induces a loss of gap junction plaques and a decrease in the phosphorylated gap junction protein connexin43-P2 (Cx43-P2), which is associated with the plaques. In this study we have considered several mechanisms. The loss of junctional plaques could be due to disaggregation of junctional particles or to endocytosis of the plaques, while the loss of Cx43-P2 could be due to dephosphorylation or degradation. Immunohistochemical analyses of DDT- or lindane-treated cells revealed a reduction in plasma membranous Cx43-positive gap junction plaques coincident with the appearance of Cx43-positive punctate cytoplasmic structures. The cytoplasmic Cx43-positive structures eventually disappeared after 4 h treatment. Diffuse Cx43-positive plasma membranous staining was not seen following DDT or lindane treatment. Western blot analyses of these cells indicated that Cx43-P2 decreased in a time-dependent manner that paralleled the disappearance of gap junction plaques from the plasma membrane. The loss of Cx43-P2 was not due to dephosphorylation, since no increase in non-phosphorylated (Cx43-NP) or other phosphorylated (Cx43-P1) forms of the protein were evident. The decrease in Cx43-P2 and the disappearance of cytoplasmic Cx43-positive structures were prevented by colchicine and chloroquine, which suggests that Cx43-P2-containing plaques were internalized and degraded in lysosomes. In addition, two small (approximately 18 and approximately 22 kDa) bands appeared in Western blots coincident with the loss of Cx43-P2 and may be degradation products of the protein. These immunohistochemical and biochemical data strongly suggest that the loss of gap junction plaques and of Cx43-P2 in WB-F344 cells treated with DDT and lindane were due to endocytosis of the plaques and degradation of Cx43-P2 in lysosomes.

  4. Preosteocytes/Osteocytes Have the Potential to Dedifferentiate Becoming a Source of Osteoblasts

    PubMed Central

    Torreggiani, Elena; Matthews, Brya G.; Pejda, Slavica; Matic, Igor; Horowitz, Mark C.; Grcevic, Danka; Kalajzic, Ivo

    2013-01-01

    Presently there is no clear evidence for the ability of mature osteogenic lineage cells to dedifferentiate. In order to identify and trace mature osteogenic lineage cells, we have utilized transgenic mouse models in which the dentin matrix protein 1 (Dmp1) promoter drives expression of GFP (active marker) or Cre recombinase (historic label) in preosteocytes/osteocytes. In long bone chip outgrowth cultures, in which cells on the bone surface were enzymatically removed, cells with previous activity of the Dmp1 promoter migrated onto plastic and down-regulated Dmp1-GFP expression. Dmp1Cre-labeled cells from these cultures had the potential to re-differentiate into the osteogenic lineage, while the negative population showed evidence of adipogenesis. We observed numerous Dmp1Cre-labeled osteoblasts on the surface of bone chips following their in vivo transplantation. Our data indicate that cells embedded in bone matrix are motile, and once given access to the extra bony milieu will migrate out of their lacunae. This population of cells is phenotypically and functionally heterogeneous in vitro. Once the preosteocytes/osteocytes leave lacunae, they can dedifferentiate, potentially providing an additional source of functional osteoblasts. PMID:24040401

  5. Potential of l-thyroxine to differentiate osteoblast-like cells via Angiopoietin1.

    PubMed

    Park, See-Hyoung; Lee, Jongsung; Kang, Mi-Ae; Moon, Young Jae; Wang, Sung Il; Kim, Kyoung Min; Park, Byung-Hyun; Jang, Kyu Yun; Kim, Jung Ryul

    2016-09-23

    Angiogenesis is closely associated with osteoblast differentiation. Previously, we demonstrated that bone formation can be accelerated by treatment with COMP-Angiopoietin1, a known angiogenic factor. Angiopoietin1 (Ang1) is a specific growth factor that generates stable and mature vasculature through the Tie2 receptor. In this study, we aimed to identify a novel drug that can activate endogenous Ang1 expression as a pharmacological treatment for bone formation. Therefore, Ang1 expression was examined in U2OS osteoblast-like cells treated with 770 drugs from a library of Food and Drug Administration (FDA)-approved drugs by using ELISA for Ang1. l-thyroxine was selected as a novel drug candidate. l-Thyroxine is a synthetic form of the hormone thyroxine, which is used to treat patients with hypothyroidism. Enzyme-linked immunosorbent assays (ELISAs) were performed to test whether Ang1 is induced in a dose-dependent manner in human osteoblast-like cell lines, U2OS and MG63. The effects of l-thyroxine on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and Alizarin red s staining. To determine the molecular mechanism, the expression of proteins related to bone formation and differentiation, such as type I collagen (COL1A1), osteocalcin (OC), bone sialoprotein (BSP), distal-less homeobox 5 (Dlx5), Runt-related transcription factor 2 (Runx2), osterix (OSX), and ALP, was tested by Western blotting analysis. Consequently, l-thyroxine induced Ang1 expression in a dose-dependent manner in both U2OS and M63 cells, which was confirmed by ELISA and Western blotting. Also, l-thyroxine activated ALP activity in U2OS and MG63 cells as well as ALP expression. Furthermore, l-thyroxine enhanced the expression of COL1A1, Runx2, OC, BSP, Dlx5, and OSX mRNA and proteins. Taken together, we demonstrated that l-thyroxine increased Ang1 expression and induces bone formation, differentiation, and mineralization in U2OS and MG63 cell lines

  6. Potential of l-thyroxine to differentiate osteoblast-like cells via Angiopoietin1.

    PubMed

    Park, See-Hyoung; Lee, Jongsung; Kang, Mi-Ae; Moon, Young Jae; Wang, Sung Il; Kim, Kyoung Min; Park, Byung-Hyun; Jang, Kyu Yun; Kim, Jung Ryul

    2016-09-23

    Angiogenesis is closely associated with osteoblast differentiation. Previously, we demonstrated that bone formation can be accelerated by treatment with COMP-Angiopoietin1, a known angiogenic factor. Angiopoietin1 (Ang1) is a specific growth factor that generates stable and mature vasculature through the Tie2 receptor. In this study, we aimed to identify a novel drug that can activate endogenous Ang1 expression as a pharmacological treatment for bone formation. Therefore, Ang1 expression was examined in U2OS osteoblast-like cells treated with 770 drugs from a library of Food and Drug Administration (FDA)-approved drugs by using ELISA for Ang1. l-thyroxine was selected as a novel drug candidate. l-Thyroxine is a synthetic form of the hormone thyroxine, which is used to treat patients with hypothyroidism. Enzyme-linked immunosorbent assays (ELISAs) were performed to test whether Ang1 is induced in a dose-dependent manner in human osteoblast-like cell lines, U2OS and MG63. The effects of l-thyroxine on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and Alizarin red s staining. To determine the molecular mechanism, the expression of proteins related to bone formation and differentiation, such as type I collagen (COL1A1), osteocalcin (OC), bone sialoprotein (BSP), distal-less homeobox 5 (Dlx5), Runt-related transcription factor 2 (Runx2), osterix (OSX), and ALP, was tested by Western blotting analysis. Consequently, l-thyroxine induced Ang1 expression in a dose-dependent manner in both U2OS and M63 cells, which was confirmed by ELISA and Western blotting. Also, l-thyroxine activated ALP activity in U2OS and MG63 cells as well as ALP expression. Furthermore, l-thyroxine enhanced the expression of COL1A1, Runx2, OC, BSP, Dlx5, and OSX mRNA and proteins. Taken together, we demonstrated that l-thyroxine increased Ang1 expression and induces bone formation, differentiation, and mineralization in U2OS and MG63 cell lines

  7. Characterization of mesenchymal stem cell subpopulations from human amniotic membrane with dissimilar osteoblastic potential.

    PubMed

    Leyva-Leyva, Margarita; Barrera, Lourdes; López-Camarillo, César; Arriaga-Pizano, Lourdes; Orozco-Hoyuela, Gabriel; Carrillo-Casas, Erika M; Calderón-Pérez, Jaime; López-Díaz, Annia; Hernandez-Aguilar, Felipe; González-Ramírez, Ricardo; Kawa, Simón; Chimal-Monroy, Jesús; Fuentes-Mera, Lizeth

    2013-04-15

    Human fetal mesenchymal stem cells can be isolated from the amniotic membrane (AM-hMSCs) by enzymatic digestion. The biological properties of this cell population have been characterized; however, few studies have focused on the presence of stem cell subpopulations and their differentiation potential. The aim of the present study was to isolate homogeneous AM-hMSC subpopulations based on the coexpression of surface markers. In addition, we aimed to characterize stem cell subpopulations through the detection of typical stem cell markers and its differentiation potential. In this study, fluorescence-activated cell sorting (FACS) was used to positively select for the surface markers CD44, CD73, and CD105. Two subpopulations were isolated: CD44+ / CD73+ / CD105+ (CD105+), and CD44+ / CD73+ / CD105- (CD105-). To characterize the cell subpopulations, the expression of pluripotency-associated markers was analyzed by reverse transcriptase-polymerase chain reaction and immunofluorescence. Our results showed positive expression of SOX2, SOX3, PAX6, OCT3/4, and NANOG in the CD105+ and CD105(-) cell subpopulations. In contrast, we did not detect expression of SSEA4 or FOXD3 in either subpopulation. Immunophenotypes, such as mesenchymal and hematopoietic markers, were studied by FACS analyses. Our data revealed the expression of the CD49a, CD49d, CD29, integrin α9β1, CD44, CD73, and CD105 antigens in both subpopulations. In contrast, CD90, CD45, CD34, CD14, and HLA-DR expression was not detected. The ability of both subpopulations to differentiate into osteoblasts, adipocytes, and chondrocytes was evidenced using Alizarin red, Oil-Red, and Alcian blue staining, respectively. Furthermore, neuronal differentiation was demonstrated by the expression of GFAP and NEURO-D. Interestingly, we observed a dissimilar osteoblastic differentiation potential between the subpopulations. CD105- cells showed stronger expression of secreted protein acidic and rich in cysteine (SPARC) and

  8. Localization and osteoblastic differentiation potential of neural crest-derived cells in oral tissues of adult mice.

    PubMed

    Ono, Miki; Suzawa, Tetsuo; Takami, Masamichi; Yamamoto, Gou; Hosono, Tomohiko; Yamada, Atsushi; Suzuki, Dai; Yoshimura, Kentaro; Watahiki, Junichi; Hayashi, Ryuhei; Arata, Satoru; Mishima, Kenji; Nishida, Kohji; Osumi, Noriko; Maki, Koutaro; Kamijo, Ryutaro

    2015-09-01

    In embryos, neural crest cells emerge from the dorsal region of the fusing neural tube and migrate throughout tissues to differentiate into various types of cells including osteoblasts. In adults, subsets of neural crest-derived cells (NCDCs) reside as stem cells and are considered to be useful cell sources for regenerative medicine strategies. Numerous studies have suggested that stem cells with a neural crest origin persist into adulthood, especially those within the mammalian craniofacial compartment. However, their distribution as well as capacity to differentiate into osteoblasts in adults is not fully understood. To analyze the precise distribution and characteristics of NCDCs in adult oral tissues, we utilized an established line of double transgenic (P0-Cre/CAG-CAT-EGFP) mice in which NCDCs express green fluorescent protein (GFP) throughout their life. GFP-positive cells were scattered like islands throughout tissues of the palate, gingiva, tongue, and buccal mucosa in adult mice, with those isolated from the latter shown to form spheres, typical cell clusters composed of stem cells, under low-adherent conditions. Furthermore, GFP-positive cells had markedly increased alkaline phosphatase (a marker enzyme of osteoblast differentiation) activity and mineralization as shown by alizarin red staining, in the presence of bone morphogenetic protein (BMP)-2. These results suggest that NCDCs reside in various adult oral tissues and possess potential to differentiate into osteoblastic cells. NCDCs in adults may be a useful cell source for bone regeneration strategies.

  9. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  10. Differential sensitivity of osteoblasts and bacterial pathogens to 405-nm light highlighting potential for decontamination applications in orthopedic surgery

    NASA Astrophysics Data System (ADS)

    Ramakrishnan, Praveen; Maclean, Michelle; MacGregor, Scott J.; Anderson, John G.; Grant, M. Helen

    2014-10-01

    Healthcare associated infections pose a major threat to patients admitted to hospitals and infection rates following orthopedic arthroplasty surgery are as high as 4%. A 405-nm high-intensity narrow spectrum light has been proven to reduce environmental contamination in hospital isolation rooms, and there is potential to develop this technology for application in arthroplasty surgery. Cultured rat osteoblasts were exposed to varying light intensities and it was found that exposures of up to a dose of 36 J/cm2 had no significant effect on cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], function (alkaline phosphatase activity), and proliferation rate (BrdU cell proliferation assay). High irradiance exposures (54 J/cm2) significantly affected the cell viability indicating that the effects of 405-nm light on osteoblasts are dose dependent. Additionally, exposure of a variety of clinically related bacteria to a dose of 36 J/cm2 resulted in up to 100% kill. These results demonstrating the differential sensitivity of osteoblasts and bacteria to 405-nm light are an essential step toward developing the technique for decontamination in orthopedic surgery.

  11. 405 nm Light exposure of osteoblasts and inactivation of bacterial isolates from arthroplasty patients: potential for new disinfection applications?

    PubMed

    McDonald, R S; Gupta, S; Maclean, M; Ramakrishnan, P; Anderson, J G; MacGregor, S J; Meek, R M D; Grant, M H

    2013-01-01

    Infection rates after arthroplasty surgery are between 1-4 %, rising significantly after revision procedures. To reduce the associated costs of treating these infections, and the patients' post-operative discomfort and trauma, a new preventative method is required. High intensity narrow spectrum (HINS) 405 nm light has bactericidal effects on a wide range of medically important bacteria, and it reduced bacterial bioburden when used as an environmental disinfection method in a Medical Burns Unit. To prove its safety for use for environmental disinfection in orthopaedic theatres during surgery, cultured osteoblasts were exposed to HINS-light of intensities up to 15 mW/cm2 for 1 h (54 J/cm2). Intensities of up to 5 mW/cm2 for 1 h had no effect on cell morphology, activity of alkaline phosphatase, synthesis of collagen or osteocalcin expression, demonstrating that under these conditions this dose is the maximum safe exposure for osteoblasts; after exposure to 15 mW/cm2 all parameters of osteoblast function were significantly decreased. Viability (measured by protein content and Crystal Violet staining) of the osteoblasts was not influenced by exposure to 5 mW/cm2 for at least 2 h. At 5 mW/cm2 HINS-light is an effective bactericide. It killed 98.1 % of Staphylococcus aureus and 83.2 % Staphylococcus epidermis populations seeded on agar surfaces, and is active against both laboratory strains and clinical isolates from infected hip and knee arthroplasties. HINS-light could have potential for development as a method of disinfection to reduce transmission of bacteria during arthroplasty, with wider applications in diverse surgical procedures involving implantation of a medical device. PMID:23471732

  12. [Dynamics of local expression of connexin-43 and basic fibroblast growth factor receptors in patients with skin and soft-tissue infections against the background of diabetes mellitus type II].

    PubMed

    Vinnik, Iu S; Salmina, A B; Tepliakova, O V; Drobushevskaia, A I; Malinovskaia, N A; Pozhilenkova, E A; Morgun, A V; Gitlina, A G

    2014-01-01

    Clinical results of wound healing dynamics were studied in 60 patients with soft-tissue infection against the background of diabetes mellitus type II. At the same time the study considered indices of intercellular contacts protein tissue expression such as connexin 43 (Cx43) and basic fibroblast growth factor receptors (bFGFR). The basic therapy of biopsy material of wound borders was applied. The reduction of bFGFR expression and the minor growth of Cx43 expression were observed. The pain syndrome proceeded for a long time and there were signs of perifocal inflammation, retard wound healing with granulation tissue. The application of combined method of ozone therapy which included autohemotherapy with ozone and an external management of wound by ozone-oxygen mixture facilitated to considerable shortening of inflammatory phase and regeneration. It was associated with increased Cx43 expression (in 1.9 times) in comparison with initial level and bFGFR was enlarged in 1.7 times to eighth day of postoperative period.

  13. Epidermal growth factor stimulates the disruption of gap junctional communication and connexin43 phosphorylation independent of 12-0-tetradecanoylphorbol 13-acetate-sensitive protein kinase C: the possible involvement of mitogen-activated protein kinase.

    PubMed

    Kanemitsu, M Y; Lau, A F

    1993-08-01

    We previously reported that epidermal growth factor (EGF) induced the disruption of gap junctional communication (gjc) and serine phosphorylation of connexin43 (Cx43) in T51B rat liver epithelial cells. However, the cascade of events linking EGF receptor activation to these particular responses have not been fully characterized. Furthermore, the serine kinase(s) acting directly on Cx43 remain unidentified. In the current study, we demonstrate that downmodulation of 12-0-tetradecanoylphorbol 13-acetate (TPA)-sensitive protein kinase C (PKC) activity does not affect EGF's ability to reduce junctional permeability or phosphorylate Cx43 in T51B cells. EGF in the presence or absence of chronic TPA treatment stimulated marked increases in Cx43 phosphorylation on numerous sites as determined by two-dimensional tryptic phosphopeptide mapping. Computer-assisted sequence analysis of Cx43 identified several protein kinase phosphorylation consensus sites including two sites for mitogen-activated protein (MAP) kinase. EGF stimulated activation of MAP kinase in a time- and dose-dependent manner where the kinetics of kinase activity corroborated its possible involvement in mediating EGF's effects. Moreover, purified MAP kinase directly phosphorylated Cx43 on serine residues in vitro. Two-dimensional tryptic and chymotryptic phosphopeptide mapping demonstrated that the in vitro phosphopeptides represented a specific subset of the in vivo phosphopeptides produced in response to EGF after chronic TPA treatment. Therefore, EGF-induced disruption of gjc and phosphorylation of Cx43 may be mediated in part by MAP kinase in vivo.

  14. Potential of Resveratrol Analogues as Antagonists of Osteoclasts and Promoters of Osteoblasts

    PubMed Central

    Boissy, Patrice; Abdallah, Basem M.; Hansen, Frederik Dagnaes; Erben, Reinhold G.; Savouret, Jean-Francois; Søe, Kent; Andersen, Thomas L.; Plesner, Torben; Delaisse, Jean-Marie

    2010-01-01

    The plant phytoalexin resveratrol was previously demonstrated to inhibit the differentiation and bone resorbing activity of osteoclasts, to promote the formation of osteoblasts from mesenchymal precursors in cultures, and inhibit myeloma cell proliferation, when used at high concentrations. In the current study, we screened five structurally modified resveratrol analogues for their ability to modify the differentiation of osteoclasts and osteoblasts and proliferation of myeloma cells. Compared to resveratrol, analogues showed an up to 5,000-fold increased potency to inhibit osteoclast differentiation. To a lesser extent, resveratrol analogues also promoted osteoblast maturation. However, they did not antagonize the proliferation of myeloma cells. The potency of the best-performing candidate in vitro was tested in vivo in an ovariectomy-induced model of osteoporosis, but an effect on bone loss could not be detected. Based on their powerful antiresorptive activity in vitro, resveratrol analogues might be attractive modulators of bone remodeling. However, further studies are required to establish their efficacy in vivo. Electronic supplementary material The online version of this article (doi:10.1007/s00223-010-9399-3) contains supplementary material, which is available to authorized users. PMID:20842496

  15. The TLR3 Ligand polyI:C Downregulates Connexin 43 Expression and Function in Astrocytes by a Mechanism Involving the NF-κB and PI3 Kinase Pathways

    PubMed Central

    ZHAO, YONGMEI; RIVIECCIO, MARK A.; LUTZ, SARAH; SCEMES, ELIANA; BROSNAN, CELIA F.

    2009-01-01

    Toll-like receptor 3 (TLR3) is a component of the innate immune response that responds to dsRNA viruses and virus replication intermediates. In this study we show that activation of astrocytes with the dsRNA mimetic polyinosinic-cytidylic acid (pI:C) results in loss of expression of connexin43 (Cx43) mRNA and protein while upregulating the expression of the ionotropic P2 receptor P2X4R. Analysis of the signaling pathways involved failed to demonstrate a role for the p38 MAP kinase, ERK, or JNK signaling pathways whereas an inhibitor of the PI3 kinase/Akt pathway effectively blocked the action of pI:C. Using adenoviral vectors containing a super-repressor of NF-κB (NF-κB SR) construct or a dominant negative interferon regulatory factor 3 (dnIRF3) construct showed that inhibition of both transcription factors also blocked the effects of pI:C. To explore the functional consequences of pI:C activation we used a pore-forming assay for P2X4R activity and a scrape loading assay for gap junction intercellular communication (GJIC). No pore-forming activity consistent with functional P2X4R expression was detected in either control or activated astrocytes. In contrast, robust Lucifer yellow transfer indicative of GJIC was detected in resting cells that was lost following pI:C activation. The dnIRF3 construct failed to restore GJIC whereas the NF-κB SR or the NF-κB inhibitor BAY11-7082 and the PI3K inhibitor LY294002 all significantly reversed the effect of pI:C on GJ connectivity. We conclude that activation of the innate immune response in astrocytes is associated with functional loss of GJIC through a pathway involving NF-κB and PI3 kinase. PMID:16958087

  16. Tyrosine-Dependent Basolateral Targeting of Human Connexin43-eYFP in Madin-Darby Canine Kidney Cells Can Be Disrupted by the Oculodentodigital Dysplasia Mutation L90V

    PubMed Central

    Chtchetinin, Jana; Gifford, Wes D.; Li, Sichen; Paznekas, William A.; Jabs, Ethylin Wang; Lai, Albert

    2009-01-01

    SUMMARY Polarized membrane sorting of Connexin 43 (Cx43) has not been well-characterized. Based on the presence of a putative sorting signal, Y286KLV289, within its carboxy terminal cytoplasmic domain, we hypothesized that Cx43 is selectively expressed on the basolateral surface of Madin-Darby canine kidney (MDCK) cells in a tyrosine-dependent manner. We generated stable MDCK cell lines expressing human wild-type and mutant Cx43-eYFP and analyzed the membrane localization of Cx43-eYFP within polarized monolayers using confocal microscopy and selective surface biotinylation. We found that wild-type Cx43-eYFP was selectively targeted to the basolateral membrane domain of MDCK cells. Substitution of alanine for Y286 disrupted basolateral targeting of Cx43-eYFP. Additionally, transplantation of a sequence containing the transferrin receptor internalization signal, LSYTRF, for P284GYKLV289 also disrupted basolateral targeting. Taken together, these results indicate that Y286 in its native amino acid sequence is necessary for targeting Cx43-eYFP to the basolateral membrane domain of MDCK cells. To determine whether the F52dup or L90V oculodentodigital dysplasia (ODDD)-associated mutations could affect polarized sorting of Cx43-eYFP, we analyzed the expression of these Cx43-eYFP mutant constructs and found that the L90V mutation disrupted basolateral expression. These findings raise the possibility that alteration of polarized targeting of Cx43 by some ODDD- associated mutations may have a phenotypic contribution. PMID:19860828

  17. The Complex of Ciliary Neurotrophic Factor-Ciliary Neurotrophic Factor Receptor α Up-Regulates Connexin43 and Intercellular Coupling in Astrocytes via the Janus Tyrosine Kinase/Signal Transducer and Activator of Transcription PathwayD⃞

    PubMed Central

    Ozog, Mark A.; Bernier, Suzanne M.; Bates, Dave C.; Chatterjee, Bishwanath; Lo, Cecilia W.; Naus, Christian C.G.

    2004-01-01

    Cytokines regulate numerous cell processes, including connexin expression and gap junctional coupling. In this study, we examined the effect of ciliary neurotrophic factor (CNTF) on connexin43 (Cx43) expression and intercellular coupling in astrocytes. Murine cortical astrocytes matured in vitro were treated with CNTF (20 ng/ml), soluble ciliary neurotrophic factor receptor α (CNTFRα) (200 ng/ml), or CNTF-CNTFRα. Although CNTF and CNTFRα alone had no effect on Cx43 expression, the heterodimer CNTF-CNTFRα significantly increased both Cx43 mRNA and protein levels. Cx43 immunostaining correlated with increased intercellular coupling as determined by dye transfer analysis. By using the pharmacological inhibitor α-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG490), the increase in Cx43 was found to be dependent on the Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Immunocytochemical analysis revealed that CNTF-CNTFRα treatment produced nuclear localization of phosphorylated STAT3, whereas CNTF treatment alone did not. Transient transfection of constructs containing various sequences of the Cx43 promoter tagged to a LacZ reporter into ROS 17/2.8 cells confirmed that the promoter region between -838 to -1693 was deemed necessary for CNTF-CNTFRα to induce heightened expression. CNTF-CNTFRα did not alter Cx30 mRNA levels, suggesting selectivity of CNTF-CNTFRα for connexin signaling. Together in the presence of soluble receptor, CNTF activates the JAK/STAT pathway leading to enhanced Cx43 expression and intercellular coupling. PMID:15342787

  18. Effects of aluminum oxide (Al2O3) nanoparticles on ECG, myocardial inflammatory cytokines, redox state, and connexin 43 and lipid profile in rats: possible cardioprotective effect of gallic acid.

    PubMed

    El-Hussainy, El-Hussainy M A; Hussein, Abdelaziz M; Abdel-Aziz, Azza; El-Mehasseb, Ibrahim

    2016-08-01

    The objectives of present study were to examine the effects of aluminum oxide (Al2O3) nanoparticles on myocardial functions, electrical activities, morphology, inflammation, redox state, and myocardial expression of connexin 43 (Cx43) and the effect of gallic acid (GA) on these effects in a rat animal model. Forty male albino rats were divided into 4 equal groups: the control (normal) group; the Al2O3 group, rats received Al2O3 (30 mg·kg(-1), i.p.) daily for 14 days; the nano-alumina group, rats received nano-alumina (30 mg·kg(-1), i.p.) daily for 14 days; and the nano-alumina + GA group, rats received GA (100 mg·kg(-1) orally once daily) for 14 days before nano-alumina administration. The results showed disturbed ECG variables and significant increases in serum levels of LDH, creatine phosphokinase (CPK), CK-MB, triglycerides (TGs), cholesterol and LDL, nitric oxide (NO), and TNF-α and myocardial concentrations of NO, TNF-α, and malondialdehyde (MDA), with significant decreases in serum HDL and myocardial GSH, SOD, catalase (CAT), and Cx43 expression in the nano-alumina group. Pretreatment with GA improved significantly all parameters except serum and myocardial NO. We concluded that chronic administration of Al2O3 NPs caused myocardial dysfunctions, and pretreatment with GA ameliorates myocardial injury induced by nano-alumina, probably through its hypolipidaemic, anti-inflammatory, and antioxidant effects and upregulation of Cx43 in heart.

  19. Depot-specific and hypercaloric diet-induced effects on the osteoblast and adipocyte differentiation potential of adipose-derived stromal cells.

    PubMed

    Sadie-Van Gijsen, Hanel; Smith, Wayne; du Toit, Eugene Francois; Michie, John; Hough, F S; Ferris, William Frank

    2012-01-01

    Adipose-derived stromal cells (ADSCs) can be differentiated in vitro into several mesenchyme-derived cell types. We had previously described depot-specific differences in the adipocyte differentiation of ADSCs, and consequently we hypothesized that there may also be depot-specific differences in osteoblast differentiation of ADSCs. For this study, the osteoblast differentiation potential of rat subcutaneous ADSCs (scADSCs) and perirenal visceral ADSCs (pvADSCs) was compared. Osteoblast differentiation media (OM) induced markers of the osteoblastic phenotype in scADSCs, but not in pvADSCs. ADSCs harvested from rats with diet-induced visceral obesity (DIO) exhibited reduced osteoinduction, compared to lean controls, but adipocyte differentiation was not affected. Expression of the pro-osteogenic transcription factor Msx2 was significantly higher in naïve scADSCs from lean and DIO rats than in pvADSCs. Our findings indicate that ADSCs from different anatomical sites are uniquely pre-programmed in vivo in a depot-specific manner, and that diet-induced metabolic disturbances translate into reduced osteoblast differentiation of ADSCs.

  20. Suppression of PKCε-mediated mitochondrial connexin 43 phosphorylation at serine 368 is involved in myocardial mitochondrial dysfunction in a rat model of dilated cardiomyopathy.

    PubMed

    Shan, Hu; Wei, Jin; Zhang, Ming; Lin, Lin; Yan, Rui; Zhang, Rong; Zhu, Yan-He

    2015-06-01

    Mitochondrial connexin 43 (Cx43) is important in cardioprotection by ischemic preconditioning; however, whether mitochondrial Cx43 is involved in mitochondrial dysfunction in the pathogenesis of dilated cardiomyopathy (DCM) remains to be elucidated. The present study was performed to investigate the changes in expression and the phosphorylation state of mitochondrial Cx43 in a rat model of DCM, and to determine whether the altered phosphorylation state of mitochondrial Cx43 was involved in mitochondrial dysfunction. A rat model of DCM was generated by daily oral administration of furazolidone (FZD) for 30 weeks. Reverse transcription polymerase chain reaction and western blot analysis revealed a decrease in the overall expression of Cx43, accompanied by reduced levels of serine 368‑phosphorylated‑Cx43 immunoreactivity in the myocardium and myocardial mitochondria. In addition, the mitochondrial membrane potential and the activities of cytochrome c oxidase, succinate dehydrogenase and protein kinase C (PKC) ε were all significantly reduced compared with those of the control group. Phorbol‑12‑myristate‑13‑acetate (PMA), a specific PKC activator, partially reversed the FZD‑induced mitochondrial Cx43 dephosphorylation at serine 368 and mitochondrial dysfunction in the cardiomyocytes. However, pretreatment with 18β‑glycerrhetinic acid, a connexin channel inhibitor, eliminated the mitochondrial protective effect of PMA in the cardiomyocytes sparsely plated without cell to cell contact. These results suggested that dephosphorylation of mitochondrial Cx43 at serine 368, due to the suppression of PKCε activity, may be a novel mechanism for mitochondrial dysfunction in the pathogenesis of DCM. PMID:25625661

  1. Amitriptyline up-regulates connexin43-gap junction in rat cultured cortical astrocytes via activation of the p38 and c-Fos/AP-1 signalling pathway

    PubMed Central

    Morioka, N; Suekama, K; Zhang, F F; Kajitani, N; Hisaoka-Nakashima, K; Takebayashi, M; Nakata, Y

    2014-01-01

    Background and Purpose Intercellular communication via gap junctions, comprised of connexin (Cx) proteins, allow for communication between astrocytes, which in turn is crucial for maintaining CNS homeostasis. The expression of Cx43 is decreased in post-mortem brains from patients with major depression. A potentially novel mechanism of tricyclic antidepressants is to increase the expression and functioning of gap junctions in astrocytes. Experimental Approach The effect of amitriptyline on the expression of Cx43 and gap junction intercellular communication (GJIC) in rat primary cultured cortical astrocytes was investigated. We also investigated the role of p38 MAPK intracellular signalling pathway in the amitriptyline-induced expression of Cx43 and GJIC. Key Results Treatment with amitriptyline for 48 h significantly up-regulated Cx43 mRNA, protein and GJIC. The up-regulation of Cx43 was not monoamine-related since noradrenaline, 5-HT and dopamine did not induce Cx43 expression and pretreatment with α- and β-adrenoceptor antagonists had no effect. Intracellular signalling involved p38 MAPK, as amitriptyline significantly increased p38 MAPK phosphorylation and Cx43 expression and GJIC were significantly blocked by the p38 inhibitor SB 202190. Furthermore, amitriptyline-induced Cx43 expression and GJIC were markedly reduced by transcription factor AP-1 inhibitors (curcumin and tanshinone IIA). The translocation of c-Fos from the cytosol and the nucleus of cortical astrocytes was increased by amitriptyline, and this response was dependent on p38 activity. Conclusion and Implication These findings indicate a novel mechanism of action of amitriptyline through cortical astrocytes, and further suggest that targeting this mechanism could lead to the development of a new class of antidepressants. PMID:24641259

  2. A New Method to Investigate How Mechanical Loading of Osteocytes Controls Osteoblasts

    PubMed Central

    Vazquez, Marisol; Evans, Bronwen A. J.; Riccardi, Daniela; Evans, Sam L.; Ralphs, Jim R.; Dillingham, Christopher Mark; Mason, Deborah J.

    2014-01-01

    Mechanical loading, a potent stimulator of bone formation, is governed by osteocyte regulation of osteoblasts. We developed a three-dimensional (3D) in vitro co-culture system to investigate the effect of loading on osteocyte–osteoblast interactions. MLO-Y4 cells were embedded in type I collagen gels and MC3T3-E1(14) or MG63 cells layered on top. Ethidium homodimer staining of 3D co-cultures showed 100% osteoblasts and 86% osteocytes were viable after 7 days. Microscopy revealed osteoblasts and osteocytes maintain their respective ovoid/pyriform and dendritic morphologies in 3D co-cultures. Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) of messenger ribonucleic acid (mRNA) extracted separately from osteoblasts and osteocytes, showed that podoplanin (E11), osteocalcin, and runt-related transcription factor 2 mRNAs were expressed in both cell types. Type I collagen (Col1a1) mRNA expression was higher in osteoblasts (P < 0.001), whereas, alkaline phosphatase mRNA was higher in osteocytes (P = 0.001). Immunohistochemistry revealed osteoblasts and osteocytes express E11, type I pro-collagen, and connexin 43 proteins. In preliminary experiments to assess osteogenic responses, co-cultures were treated with human recombinant bone morphogenetic protein 2 (BMP-2) or mechanical loading using a custom built loading device. BMP-2 treatment significantly increased osteoblast Col1a1 mRNA synthesis (P = 0.031) in MLO-Y4/MG63 co-cultures after 5 days treatment. A 16-well silicone plate, loaded (5 min, 10 Hz, 2.5 N) to induce 4000–4500 με cyclic compression within gels increased prostaglandin E2 (PGE2) release 0.5 h post-load in MLO-Y4 cells pre-cultured in 3D collagen gels for 48, 72 h, or 7 days. Mechanical loading of 3D co-cultures increased type I pro-collagen release 1 and 5 days later. These methods reveal a new osteocyte–osteoblast co-culture model that may be useful for investigating mechanically induced

  3. MEK5 suppresses osteoblastic differentiation

    SciTech Connect

    Kaneshiro, Shoichi; Otsuki, Dai; Yoshida, Kiyoshi; Yoshikawa, Hideki; Higuchi, Chikahisa

    2015-07-31

    Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and is activated by its upstream kinase, MAPK kinase 5 (MEK5), which is a member of the MEK family. Although the role of MEK5 has been investigated in several fields, little is known about its role in osteoblastic differentiation. In this study, we have demonstrated the role of MEK5 in osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. We found that treatment with BIX02189, an inhibitor of MEK5, increased alkaline phosphatase (ALP) activity and the gene expression of ALP, osteocalcin (OCN) and osterix, as well as it enhanced the calcification of the extracellular matrix. Moreover, osteoblastic cell proliferation decreased at a concentration of greater than 0.5 μM. In addition, knockdown of MEK5 using siRNA induced an increase in ALP activity and in the gene expression of ALP, OCN, and osterix. In contrast, overexpression of wild-type MEK5 decreased ALP activity and attenuated osteoblastic differentiation markers including ALP, OCN and osterix, but promoted cell proliferation. In summary, our results indicated that MEK5 suppressed the osteoblastic differentiation, but promoted osteoblastic cell proliferation. These results implied that MEK5 may play a pivotal role in cell signaling to modulate the differentiation and proliferation of osteoblasts. Thus, inhibition of MEK5 signaling in osteoblasts may be of potential use in the treatment of osteoporosis. - Highlights: • MEK5 inhibitor BIX02189 suppresses proliferation of osteoblasts. • MEK5 knockdown and MEK5 inhibitor promote differentiation of osteoblasts. • MEK5 overexpression inhibits differentiation of osteoblasts.

  4. Mesenchymal stem cell secretion of chemokines during differentiation into osteoblasts, and their potential role in mediating interactions with breast cancer cells.

    PubMed

    Molloy, Alan P; Martin, Fiachra T; Dwyer, Roisin M; Griffin, Tomas P; Murphy, Mary; Barry, Frank P; O'Brien, Timothy; Kerin, Michael J

    2009-01-15

    Over 70% of patients with advanced breast cancer will develop bone metastases for which there is no cure. Mesenchymal Stem Cells (MSCs) and their derivative osteoblasts are subpopulations of cells within the bone marrow environment, postulated as potential interacting targets for disseminating cancer cells because of their ability to secrete a range of chemokines. This study aimed to investigate chemokine secretion throughout MSC differentiation into osteoblasts and their effect on the breast cancer cells. Primary MSCs and osteoblast progenitors were cultured in appropriate conditions to induce differentiation into mature osteoblasts. Chemokines secreted throughout differentiation were detected using ChemiArray and ELISA. Migration of breast cancer cells in response to the bone-derived cells was quantified using Transwell inserts. Breast cancer cells were cocultured with MSCs, retrieved using magnetic beads, and changes in CCL2 expression were analyzed. MSCs secreted a range of factors including IL-6, TIMP-1 and CCL2, the range and level of which changed throughout differentiation. CCL2 secretion by MSCs increased significantly above control cells as they differentiated into mature osteoblasts (p<0.05). The bone-derived cells stimulated migration of breast cancer cells, and this was inhibited (21-50%) in the presence of a CCL2 antibody. CCL2 gene expression in breast cancer cells was upregulated following direct coculture with MSCs. The varying levels of chemokines secreted throughout MSC differentiation may play an important role in supporting tumor cell homing and progression. These results further highlight the distinct effect MSCs have on breast cancer cells and their potential importance in supporting development of metastases.

  5. Intercellular communications within the rat anterior pituitary. XVI: postnatal changes of distribution of S-100 protein positive cells, connexin 43 and LH-RH positive sites in the pars tuberalis of the rat pituitary gland. An immunohistochemical and electron microscopic study.

    PubMed

    Wada, Ikuo; Sakuma, Eisuke; Shirasawa, Nobuyuki; Wakabayashi, Kenjiro; Otsuka, Takanobu; Hattori, Kazuki; Yashiro, Takashi; Herbert, Damon C; Soji, Tsuyoshi

    2014-02-01

    The architecture of luteinizing hormone-releasing hormone (LH-RH) nerve ends and the S-100 protein containing folliculo-stellate cells forming gap junctions in the pars tuberalis is basically important in understanding the regulation of the hormone producing mechanism of anterior pituitary glands. In this study, intact male rats 5-60 days old were prepared for immunohistochemistry and electron microscopy. From immunostained sections, the S-100 containing cells in pars tuberalis were first detected on day 30 and increased in number to day 60; this was parallel to the immunohistochemical staining of gap junction protein, connexin 43. LH-RH positive sites were clearly observed on just behind the optic chiasm and on the root of pituitary stalk on day 30. On day 60, the width of layer increased, while follicles and gap junctions were frequently observed between agranular cells in 10 or more layers of pars tuberalis. In the present study, we investigated the sexual maturation of the anterior pituitary glands through the postnatal development of S-100 positive cells, connexin 43 and LH-RH nerves. It is suggested that the folliculo-stellate cell system including the LH-RH neurons in the pars tuberalis participates in the control of LH secretion along with the portal vein system. PMID:24216131

  6. Effects of pyrite bioleaching solution of Acidithiobacillus ferrooxidans on viability, differentiation and mineralization potentials of rat osteoblasts.

    PubMed

    Zhou, Jian; Chen, Ke-Ming; Zhi, De-Juan; Xie, Qin-Jian; Xian, Cory J; Li, Hong-Yu

    2015-12-01

    Iron pyrite, an important component of traditional Chinese medicine, has a poor solubility, bioavailability, and patient compliance due to a high dose required and associated side effects, all of which have limited its clinical applications and experimental studies on its action mechanisms in improving fracture healing. This study investigated Acidithiobacillus ferrooxidans (A.f)-bioleaching of two kinds of pyrites and examined bioactivities of the derived solutions in viability and osteogenic differentiation in rat calvarial osteoblasts. A.f bioleaching improved element contents (Fe, Mn, Zn, Cu, and Se) in the derived solutions and the solutions concentration-dependently affected osteoblast viability and differentiation. While the solutions had no effects at low concentrations and inhibited the osteoblast alkaline phosphatase (ALP) activity at high concentrations, they improved ALP activity at their optimal concentrations. The improved osteoblast differentiation and osteogenic function at optimal concentrations were also revealed by levels of ALP cytochemical staining, calcium deposition, numbers and areas of mineralized nodules formed, mRNA and protein expression levels of osteogenesis-related genes (osteocalcin, Bmp-2, Runx-2, and IGF-1), and Runx-2 nuclear translocation. Data from this study will be useful in offering new strategies for improving pyrite bioavailability and providing a mechanistic explanation for the beneficial effects of pyrite in improving bone healing. PMID:26283321

  7. Effects of pyrite bioleaching solution of Acidithiobacillus ferrooxidans on viability, differentiation and mineralization potentials of rat osteoblasts.

    PubMed

    Zhou, Jian; Chen, Ke-Ming; Zhi, De-Juan; Xie, Qin-Jian; Xian, Cory J; Li, Hong-Yu

    2015-12-01

    Iron pyrite, an important component of traditional Chinese medicine, has a poor solubility, bioavailability, and patient compliance due to a high dose required and associated side effects, all of which have limited its clinical applications and experimental studies on its action mechanisms in improving fracture healing. This study investigated Acidithiobacillus ferrooxidans (A.f)-bioleaching of two kinds of pyrites and examined bioactivities of the derived solutions in viability and osteogenic differentiation in rat calvarial osteoblasts. A.f bioleaching improved element contents (Fe, Mn, Zn, Cu, and Se) in the derived solutions and the solutions concentration-dependently affected osteoblast viability and differentiation. While the solutions had no effects at low concentrations and inhibited the osteoblast alkaline phosphatase (ALP) activity at high concentrations, they improved ALP activity at their optimal concentrations. The improved osteoblast differentiation and osteogenic function at optimal concentrations were also revealed by levels of ALP cytochemical staining, calcium deposition, numbers and areas of mineralized nodules formed, mRNA and protein expression levels of osteogenesis-related genes (osteocalcin, Bmp-2, Runx-2, and IGF-1), and Runx-2 nuclear translocation. Data from this study will be useful in offering new strategies for improving pyrite bioavailability and providing a mechanistic explanation for the beneficial effects of pyrite in improving bone healing.

  8. Continuous separation of cells of high osteoblastic differentiation potential from mesenchymal stem cells on an antibody-immobilized column.

    PubMed

    Mahara, Atsushi; Yamaoka, Tetsuji

    2010-05-01

    Here, we report that two distinctive cell populations with osteoblastic differentiation ability were found in adherent cell populations from bone marrow. Mesenchymal stem cells (MSCs) were conventionally isolated by using adherent property of bone marrow cells onto a plastic culture dish. MSCs enriched on the basis of their adherent property were considered phenotypically and functionally heterogeneous. We developed a ligand-immobilized surface for separating subpopulation of adherent cells derived from bone marrow by the cell rolling process. We successfully isolate two cell populations with high differentiation ability for osteoblasts in adherent bone marrow cells by using the anti-CD34 antibody-immobilized column. The antibody was covalently conjugated with polyacrylic acid and introduced onto the inner surface of a silicone tube. When cell suspension of MSCs was injected into the antibody-immobilized column, different cell populations were isolated. After the cultivation of isolated cells in the osteoblastic differentiation medium for 1 week, few sub-populations were strongly induced to form osteoblastic cells. This study revealed that the ligand-immobilized surface can be used to continually separate cell populations under a labeling-free condition. PMID:20185169

  9. Mechanical stimulation of polycystin-1 induces human osteoblastic gene expression via potentiation of the calcineurin/NFAT signalling axis

    PubMed Central

    Georgopoulou, Urania; Adamopoulos, Christos; Basdra, Efthimia K.; Papavassiliou, Athanasios G.

    2016-01-01

    Mechanical forces trigger biological responses in bone cells that ultimately control osteoblastogenesis and bone remodelling. Although several mechanosensors have been postulated, the precise mechanotransduction pathway remains obscure as the initial mechanosensing event has not yet been identified. Studies in kidney cells have shown that polycystin-1 (PC1), via its extracellular N-terminal part, may function as an “antenna-like” protein providing a linkage between environmental cues and their conversion into biochemical responses that regulate various cellular processes via the calcineurin/ NFAT pathway. Here we explored the involvement of PC1 in mechanical load (stretching)-induced signalling cascades that control osteoblastogenesis/bone formation. FACS and TransAM Transcription Factor ELISA analyses employing extracts from primary human osteoblast-like, PC1 expressing cells subjected to mechanical stretching (0-6 h) revealed that unphosphorylated/DNA-binding competent NFATc1 increased at 0.5-1 h and returned to normal at 6 h. In accordance with the activation mechanism of NFATc1, stretching of cultured cells pre-treated with cyclosporin A (CsA, a specific inhibitor of the calcineurin/NFAT pathway) abrogated the observed decrease in the abundance of the cytoplasmic pNFATc1 (phosphorylated/inactive) species. Furthermore, stretching of osteoblastic cells pre-treated with an antibody against the mechanosensing N-terminal part of PC1 also abrogated the observed decrease in the cytoplasmic levels of the inactive pNFATc1 species. Importantly, under similar conditions (pre-incubation of stretched cells with the inhibitory anti-PC1 antibody), the expression of the key osteoblastic, NFATc1-target gene runx2 decreased compared to untreated cells. Therefore PC1 acts as chief mechanosensing molecule that modulates osteoblastic gene transcription and hence bone-cell differentiation through the calcineurin/NFAT signalling cascade. PMID:23014991

  10. Altered expression of connexin43 and phosphorylation connexin43 in glioma tumors

    PubMed Central

    Ye, Xin-Yun; Jiang, Qiu-Hua; Hong, Tao; Zhang, Zhen-Yu; Yang, Rui-Jin; Huang, Jin-Qing; Hu, Kun; Peng, Yu-Ping

    2015-01-01

    In this study, we aim to evaluate the connexin (Cx43) and phosphorylation Cx43 (p-Cx43) expression of human glioma tumors and correlate their expression with degrees of malignancy and proliferation, apoptosis, and migration activity of tumors. Cx43 and p-Cx43 expression were examined by Western blot analysis and immunohistochemical staining. The U251 cell viability was measured by MTT analysis. The apoptosis and migration were also evaluated by flow cytometric analysis and fluoroblok transwell chambers, respectively. We found that the Cx43 expression were significantly downregulated in in malignant glioma (WHO grade III and IV), compared to the malignant glioma (WHO grade I and II) and the p-Cx43 expression levels of malignant glioma (WHO grade III and IV) were significantly increased (P<0.05), compared to the malignant glioma (WHO grade I and II) at immunohistochemical analysis. After treatment of cells with a specific inhibitor of PKC, MAPK, and PTK inhibitors, the cell viability and migration were significantly decreased, while the apoptosis was slightly induced. In conclusion, the Cx43 expression level is inversely correlated with the tumor grade and proliferation and migration activity of tumor. Higher p-Cx43 expression level in high tumor grade suggests that a complex mechanism is involved in the suppression of tumor growth by connexins. PMID:26191122

  11. Are We Economically Efficient Enough to Increase the Potential of in Vitro Proliferation of Osteoblasts by Means of Pharmacochemical Agents?

    PubMed Central

    Isyar, Mehmet; Gumustas, Seyit Ali; Yilmaz, Ibrahim; Sirin, Duygu Yasar; Tosun, Hacı Bayram; Mahirogullari, Mahir

    2016-01-01

    Background: The aim of this study was to test the necessity of using expensive and unaccesible pharmacological-chemical agents in the proliferation of bone tissue cultures and in the induction of mineralized matrix formation to increase the osteogenic effect. Methods: For this purpose, human primary cell cultures were prepared and then divided into two groups. Whereas the cells in group I were fed with an osteoblast stimulator medium containing Dulbecco’s Modified Eagle Medium (DMEM) and β-glycerophosphate, the cells in group II were fed with DMEM containing dexamethasone and 2-phospho-L-ascorbic acid trisodium salt. Both groups were evaluated in terms of viability, toxicity, and proliferation and then compared in terms of cell surface morphology through inverted light and environmental scanning electron microscopy. In addition to immunoflow cytometric analyses, the effects of alkaline phosphatase activities were evaluated using the spectrophotometric method to examine the osteoblastic activities. Costs were calculated in the currency of the European Union (Euros). The Tukey Honestly Significant Difference test was used to reach the statistical evaluation of the data after the analysis of variance. Results: It was reported that the level of the alkaline phosphates was higher in group I compared to group II. It was observed that the surface morphology quality, the number of living cells, and proliferation were higher in group II and that the results were deemed statistically significant. Conclusion: It was found that the 2-phospho-L-ascorbic acid trisodium salt and dexamethasone mixture was as effective as the expensive commercial kits on the osteogenic effect on human primary bone tissue. PMID:27708738

  12. The response of osteoblastic MC3T3-E1 cells to micro- and nano-textured, hydrophilic and bioactive titanium surfaces.

    PubMed

    Lumetti, S; Manfredi, E; Ferraris, S; Spriano, S; Passeri, G; Ghiacci, G; Macaluso, G; Galli, C

    2016-04-01

    The aim of the present work was to investigate the morphology and activity of the murine osteoblastic cell line MC3T3 on control smooth (Machined), commercially available rough (ZT) titanium discs, and on titanium samples obtained by modifying the ZT treatment protocol, and herein labelled as ZTF, ZTM and ZTFM. Cells were evaluated at SEM and immunofluorescence for morphology and cell-to-cell interactions and by MTT assay and real time PCR for cell growth and function. Microscopy showed that ZT modified protocols could differently affect cell shape and distribution. All the tested surfaces showed good biocompatibility by viability assay. However, cells on smoother surfaces appeared to express higher levels of transcript for Collagen 1a1, the main component of extracellular matrix, by real time PCR. Expression of the early differentiation marker Alkaline Phosphatase was higher on ZTF surfaces and ZTM enhanced the expression of later osteoblastic markers Osteoprotegerin and Osteocalcin. Noteworthy, the expression of Connexin 43, a component of cell-to-cell contacts and hemichannels, followed a similar pattern to differentiation marker genes and was higher in cells on ZTM surfaces, consistently with the microscopic observation of cell clusters. Taken together, this data showed that ZTF and ZTM treatment protocols appeared to improve the basal sand-blasting/acid-etching ZT procedure with ZTM surfaces promoting the most mature stage of differentiation.

  13. An atmospheric-pressure plasma-treated titanium surface potentially supports initial cell adhesion, growth, and differentiation of cultured human prenatal-derived osteoblastic cells.

    PubMed

    Kawase, Tomoyuki; Tanaka, Takaaki; Minbu, Hiromi; Kamiya, Mana; Oda, Masafumi; Hara, Toshiaki

    2014-08-01

    An atmospheric-pressure plasma (APP) treatment was recently reported to render titanium (Ti) surfaces more suitable for osteoblastic cell proliferation and osteogenesis. However, the mechanism of action remains to be clearly demonstrated. In this study, we focused on cell adhesion and examined the effects of the APP treatment on the initial responses of human prenatal-derived osteoblastic cells incubated on chemically polished commercially pure Ti (CP-cpTi) plates. In the medium containing 1% fetal bovine serum, the initial cell adhesion and the actin polymerization were evaluated by scanning electron microscopy and fluorescence microscopy. The expression of cell adhesion-related molecules and osteoblast markers at the messenger RNA level was assessed by real-time quantitative polymerase chain reaction. Although the cells on the APP-treated CP-cpTi surface developed fewer cytoskeletal actin fibers, they attached with higher affinity and consequently proliferated more actively (1.46-fold over control at 72 h). However, most of the cell adhesion molecule genes were significantly downregulated (from 40 to 85% of control) in the cells incubated on the APP-treated CP-cpTi surface at 24 h. Similarly, the osteoblast marker genes were significantly downregulated (from 49 to 63% of control) at 72 h. However, the osteoblast marker genes were drastically upregulated (from 197 to 296% of control) in these cells by dexamethasone and β-glycerophosphate treatment. These findings suggest that the APP treatment improves the ability of the CP-cpTi surface to support osteoblastic proliferation by enhancing the initial cell adhesion and supports osteoblastic differentiation when immature osteoblasts begin the differentiation process.

  14. Aerobic Glycolysis in Osteoblasts

    PubMed Central

    Esen, Emel; Long, Fanxin

    2014-01-01

    Osteoblasts, the chief bone-making cells in the body, are a focus of osteoporosis research. Although teriparatite, a synthetic fragment of the human parathyroid hormone (PTH), has been an effective bone anabolic drug, there remains a clinical need for additional therapeutics that safely stimulates osteoblast number and function. Work in the past several decades has provided unprecedented clarity about the roles of growth factors and transcription factors in regulating osteoblast differentiation and activity, but whether these factors may regulate cellular metabolism to influence cell fate and function has been largely unexplored. The past few years have witnessed a resurgence of interest in the cellular metabolism of osteoblasts, with the hope that elucidation of their metabolic profile may open new avenues for developing bone anabolic agents. Here we review the current understanding about glucose metabolism in osteoblasts. PMID:25200872

  15. Investigation of low-level laser therapy potentiality on proliferation and differentiation of human osteoblast-like cells in the absence/presence of osteogenic factors

    NASA Astrophysics Data System (ADS)

    Bloise, Nora; Ceccarelli, Gabriele; Minzioni, Paolo; Vercellino, Marco; Benedetti, Laura; De Angelis, Maria Gabriella Cusella; Imbriani, Marcello; Visai, Livia

    2013-12-01

    Several studies have shown that low-level laser irradiation (LLLI) has beneficial effects on bone regeneration. The objective of this study was to examine the in vitro effects of LLLI on proliferation and differentiation of a human osteoblast-like cell line (Saos-2 cell line). Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (659 nm 10 mW power output). The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated once or for three consecutive days with laser doses of 1 or 3 J/cm2. The obtained results showed that laser stimulation enhances the proliferation potential of Saos-2 cells without changing their telomerase pattern or morphological characteristics. The effects on cell differentiation were assessed after three consecutive laser irradiation treatments in the presence or absence of osteo-inductive factors on day 14. Enhanced secretion of proteins specific for differentiation toward bone as well as calcium deposition and alkaline phosphatase activity were observed in irradiated cells cultured in a medium not supplemented with osteogenic factors. Taken together these findings indicate that laser treatment enhances the in vitro proliferation of Saos-2 cells, and also influences their osteogenic maturation, which suggest it is a helpful application for bone tissue regeneration.

  16. Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin.

    PubMed

    Knopf, Franziska; Hammond, Christina; Chekuru, Avinash; Kurth, Thomas; Hans, Stefan; Weber, Christopher W; Mahatma, Gina; Fisher, Shannon; Brand, Michael; Schulte-Merker, Stefan; Weidinger, Gilbert

    2011-05-17

    While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair. PMID:21571227

  17. Connexin43 regulates joint location in zebrafish fins.

    PubMed

    Sims, Kenneth; Eble, Diane M; Iovine, M Kathryn

    2009-03-15

    Joints are essential for skeletal form and function, yet their development remains poorly understood. In zebrafish fins, joints form between the bony fin ray segments providing essentially unlimited opportunities to evaluate joint morphogenesis. Mutations in cx43 cause the short segment phenotype of short fin (sof(b123)) mutants, suggesting that direct cell-cell communication may regulate joint location. Interestingly, increased cx43 expression in the another long fin (alf(dty86)) mutant appears to cause joint failure typical of that mutant. Indeed, knockdown of cx43 in alf(dty86) mutant fins rescues joint formation. Together, these data reveal a correlation between the level of Cx43 expression in the fin ray mesenchyme and the location of joints. Cx43 was also observed laterally in cells associated with developing joints. Confocal microscopy revealed that the Cx43 protein initially surrounds the membranes of ZNS5-positive joint cells, but at later stages becomes polarized toward the underlying Cx43-positive mesenchymal cells. One possibility is that communication between the Cx43-positive mesenchyme and the overlying ZNS5-positive cells regulates joint location, and upregulation of Cx43 in joint-forming cells contributes to joint morphogenesis. PMID:19150347

  18. Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia

    PubMed Central

    Longchamp, Alban; Allagnat, Florent; Alonso, Florian; Kuppler, Christopher; Dubuis, Céline; Ozaki, Charles-Keith; Mitchell, James R.; Berceli, Scott; Corpataux, Jean-Marc

    2015-01-01

    Venous bypass grafts often fail following arterial implantation due to excessive smooth muscle cells (VSMC) proliferation and consequent intimal hyperplasia (IH). Intercellular communication mediated by Connexins (Cx) regulates differentiation, growth and proliferation in various cell types. Microarray analysis of vein grafts in a model of bilateral rabbit jugular vein graft revealed Cx43 as an early upregulated gene. Additional experiments conducted using an ex-vivo human saphenous veins perfusion system (EVPS) confirmed that Cx43 was rapidly increased in human veins subjected ex-vivo to arterial hemodynamics. Cx43 knock-down by RNA interference, or adenoviral-mediated overexpression, respectively inhibited or stimulated the proliferation of primary human VSMC in vitro. Furthermore, Cx blockade with carbenoxolone or the specific Cx43 inhibitory peptide 43gap26 prevented the burst in myointimal proliferation and IH formation in human saphenous veins. Our data demonstrated that Cx43 controls proliferation and the formation of IH after arterial engraftment. PMID:26398895

  19. Connexin 43-enhanced suicide gene therapy using herpesviral vectors.

    PubMed

    Marconi, P; Tamura, M; Moriuchi, S; Krisky, D M; Niranjan, A; Goins, W F; Cohen, J B; Glorioso, J C

    2000-01-01

    Tumor cell transduction with the herpes simplex virus (HSV) thymidine kinase (tk) gene and treatment with ganciclovir (GCV) is a widely studied cancer gene therapy. Connexin (Cx)-dependent gap junctions between cells facilitate the intercellular spread of TK-activated GCV, thereby creating a bystander effect that improves tumor cell killing. However, tumor cells often have reduced connexin expression, thus thwarting bystander killing and the effectiveness of TK/GCV gene therapy. To improve the effectiveness of this therapy, we compared an HSV vector (TOCX) expressing Cx43 in addition to TK with an isogenic tk vector (TOZ.1) for their abilities to induce bystander killing of Cx-positive U-87 MG human glioblastoma cells and Cx-negative L929 fibrosarcoma cells in vitro and in vivo. The results showed that low-multiplicity infection of U-87 MG cells with TOCX only minimally increased GCV-mediated cell death compared with infection by TOZ.1, consistent with the endogenous level of Cx in these cells. In contrast, bystander killing of L929 cells was markedly enhanced by vector-mediated expression of Cx. In vivo experiments in which U-87 MG cells were preinfected at low multiplicity and injected into the flanks of nude mice showed complete cures of all animals in the TOCX group following GCV treatment, whereas untreated animals uniformly formed fatal tumors. TOCX injection into U-87 MG intradermal and intracranial tumors resulted in prolonged survival of the host animals in a GCV-dependent manner. Together, these results suggest that the combination of TK and Cx may be beneficial for the treatment of human glioblastoma.

  20. Vertically, interconnected carbon nanowalls as biocompatible scaffolds for osteoblast cells

    NASA Astrophysics Data System (ADS)

    Ion, Raluca; Vizireanu, Sorin; Luculescu, Catalin; Cimpean, Anisoara; Dinescu, Gheorghe

    2016-07-01

    The response of MC3T3-E1 pre-osteoblasts to vertically aligned, interconnected carbon nanowalls prepared by plasma enhanced chemical vapor deposition on silicon substrate has been evaluated in terms of cell adhesion, viability and cell proliferation. The behavior of osteoblasts seeded on carbon nanowalls was analyzed in parallel and compared with the behavior of the cells maintained in contact with tissue culture polystyrene (TCPS). The results demonstrate that osteoblasts adhere and remain viable in the long term on carbon nanowalls. Moreover, on the investigated scaffold cell proliferation was significantly promoted, although to a lower extent than on TCPS. Overall, the successful culture of osteoblasts on carbon nanowalls coated substrate confirms the biocompatibility of this scaffold, which could have potential applications in the development of orthopedic biomaterials.

  1. Platelet-rich plasma stimulates osteoblastic differentiation in the presence of BMPs

    SciTech Connect

    Tomoyasu, Akihiro; Higashio, Kanji; Kanomata, Kazuhiro; Goto, Masaaki; Kodaira, Kunihiko; Serizawa, Hiroko; Suda, Tatsuo; Nakamura, Atsushi; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu . E-mail: katagiri@saitama-med.ac.jp

    2007-09-14

    Platelet-rich plasma (PRP) is clinically used as an autologous blood product to stimulate bone formation in vivo. In the present study, we examined the effects of PRP on proliferation and osteoblast differentiation in vitro in the presence of bone morphogenetic proteins (BMPs). PRP and its soluble fraction stimulated osteoblastic differentiation of myoblasts and osteoblastic cells in the presence of BMP-2, BMP-4, BMP-6 or BMP-7. The soluble PRP fraction stimulated osteoblastic differentiation in 3D cultures using scaffolds made of collagen or hydroxyapatite. Moreover, heparin-binding fractions obtained from serum also stimulated osteoblastic differentiation in the presence of BMP-4. These results suggested that platelets contain not only growth factors for proliferation but also novel potentiator(s) for BMP-dependent osteoblastic differentiation.

  2. Osteoblast adhesion on nanophase ceramics.

    PubMed

    Webster, T J; Siegel, R W; Bizios, R

    1999-07-01

    Osteoblast adhesion on nanophase alumina (Al2O3) and titania (TiO2) was investigated in vitro. Osteoblast adhesion to nanophase alumina and titania in the absence of serum from Dulbecco's modified Eagle medium (DMEM) was significantly (P < 0.01) less than osteoblast adhesion to alumina and titania in the presence of serum. In the presence of 10% fetal bovine serum in DMEM osteoblast adhesion on nanophase alumina (23 nm grain size) and titania (32 nm grain size) was significantly (P < 0.05) greater than on conventional alumina (177 nm grain size) and titania (2.12 microm grain size), respectively, after 1, 2, and 4 h. Further investigation of the dependence of osteoblast adhesion on alumina and titania grain size indicated the presence of a critical grain size for osteoblast adhesion between 49 and 67 nm for alumina and 32 and 56 nm for titania. The present study provides evidence of the ability of nanophase alumina and titania to simulate material characteristics (such as surface grain size) of physiological bone that enhance protein interactions (such as adsorption, configuration, bioactivity, etc.) and subsequent osteoblast adhesion.

  3. Nacre extract restores the mineralization capacity of subchondral osteoarthritis osteoblasts.

    PubMed

    Brion, A; Zhang, G; Dossot, M; Moby, V; Dumas, D; Hupont, S; Piet, M H; Bianchi, A; Mainard, D; Galois, L; Gillet, P; Rousseau, M

    2015-12-01

    Osteoarthritis (OA) is the most common cause of joint chronic pain and involves the entire joints. Subchondral osteoarthritic osteoblasts present a mineralization defect and, to date, only a few molecules (Vitamin D3 and Bone Morphogenetic Protein2) could improve the mineralization potential of this cell type. In this context, we have tested for the first time the effect of nacre extract on the mineralization capacity of osteoblasts from OA patients. Nacre extract is known to contain osteogenic molecules which have demonstrated their activities notably on the MC3T3 pre-osteoblastic cell line. For this goal, molecules were extracted from nacre (ESM, Ethanol Soluble Matrix) and tested on osteoblasts of the subchondral bone from OA patients undergoing total knee replacement and on MC3T3 cells for comparison. We chose to investigate the mineralization with Alizarin Red staining and with the study of extracellular matrix (ECM) structure and composition. In a complementary way the structure of the ECM secreted during the mineralization phase was investigated using second harmonic generation (SHG). Nacre extract was able to induce the early presence (after 7 days) of precipitated calcium in cells. Raman spectroscopy and electron microscopy showed the presence of nanograins of an early crystalline form of calcium phosphate in OA osteoblasts ECM and hydroxyapatite in MC3T3 ECM. SHG collagen fibers signal was present in both cell types but lower for OA osteoblasts. In conclusion, nacre extract was able to rapidly restore the mineralization capacity of osteoarthritis osteoblasts, therefore confirming the potential of nacre as a source of osteogenic compounds.

  4. Osteoblast Differentiation at a Glance

    PubMed Central

    Rutkovskiy, Arkady; Stensløkken, Kåre-Olav; Vaage, Ingvar Jarle

    2016-01-01

    Ossification is a tightly regulated process, performed by specialized cells called osteoblasts. Dysregulation of this process may cause inadequate or excessive mineralization of bones or ectopic calcification, all of which have grave consequences for human health. Understanding osteoblast biology may help to treat diseases such as osteogenesis imperfecta, calcific heart valve disease, osteoporosis, and many others. Osteoblasts are bone-building cells of mesenchymal origin; they differentiate from mesenchymal progenitors, either directly or via an osteochondroprogenitor. The direct pathway is typical for intramembranous ossification of the skull and clavicles, while the latter is a hallmark of endochondral ossification of the axial skeleton and limbs. The pathways merge at the level of preosteoblasts, which progress through 3 stages: proliferation, matrix maturation, and mineralization. Osteoblasts can also differentiate into osteocytes, which are stellate cells populating narrow interconnecting passages within the bone matrix. The key molecular switch in the commitment of mesenchymal progenitors to osteoblast lineage is the transcription factor cbfa/runx2, which has multiple upstream regulators and a wide variety of targets. Upstream is the Wnt/Notch system, Sox9, Msx2, and hedgehog signaling. Cofactors of Runx2 include Osx, Atf4, and others. A few paracrine and endocrine factors serve as coactivators, in particular, bone morphogenetic proteins and parathyroid hormone. The process is further fine-tuned by vitamin D and histone deacetylases. Osteoblast differentiation is subject to regulation by physical stimuli to ensure the formation of bone adequate for structural and dynamic support of the body. Here, we provide a brief description of the various stimuli that influence osteogenesis: shear stress, compression, stretch, micro- and macrogravity, and ultrasound. A complex understanding of factors necessary for osteoblast differentiation paves a way to introduction

  5. Fibronectin regulates calvarial osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Moursi, A. M.; Damsky, C. H.; Lull, J.; Zimmerman, D.; Doty, S. B.; Aota, S.; Globus, R. K.

    1996-01-01

    The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not

  6. 'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells.

    PubMed

    Okamoto, Kazuma; Miyoshi, Shunichiro; Toyoda, Masashi; Hida, Naoko; Ikegami, Yukinori; Makino, Hatsune; Nishiyama, Nobuhiro; Tsuji, Hiroko; Cui, Chang-Hao; Segawa, Kaoru; Uyama, Taro; Kami, Daisuke; Miyado, Kenji; Asada, Hironori; Matsumoto, Kenji; Saito, Hirohisa; Yoshimura, Yasunori; Ogawa, Satoshi; Aeba, Ryo; Yozu, Ryohei; Umezawa, Akihiro

    2007-07-15

    The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.

  7. Inactivation of the integrin-linked kinase (ILK) in osteoblasts increases mineralization.

    PubMed

    El-Hoss, Jad; Arabian, Alice; Dedhar, Shoukat; St-Arnaud, René

    2014-01-01

    In osteoblasts, Integrin-Linked Kinase (ILK)-dependent phosphorylation of the cJUN transcriptional coactivator, αNAC, induces the nuclear accumulation of the coactivator and potentiates cJUN-dependent transcription. Mutation of the ILK phosphoacceptor site within the αNAC protein leads to cytoplasmic retention of the coactivator and cell-autonomous increases in osteoblastic activity. In order to gain further insight into the ILK-αNAC signaling cascade, we inactivated ILK using RNA knockdown in osteoblastic cells and engineered mice with specific ablation of ILK in osteoblasts. ILK knockdown in MC3T3-E1 osteoblast-like cells reduced phosphorylation of its downstream target glycogen synthase kinase 3β (GSK3β), which led to cytoplasmic retention of αNAC and increased mineralization with augmented expression of the osteoblastic differentiation markers, pro-α1(I) collagen (col1A1), Bone Sialoprotein (Bsp) and Osteocalcin (Ocn). Cultured ILK-deficient primary osteoblasts also showed increased cytoplasmic αNAC levels, and augmented mineralization with higher Runx2, Col1a1 and Bsp expression. Histomorphometric analysis of bones from mutant mice with ILK-deficient osteoblasts (Col1-Cre;Ilk(-/fl)) revealed transient changes, with increased bone volume in newborn animals that was corrected by two weeks of age. Our data suggest that the ILK-αNAC cascade acts to reduce the pace of osteoblast maturation. We propose that in vivo, functional redundancy is able to compensate for the loss of ILK activity, leading to the absence of an obvious phenotype when osteoblast-specific Ilk-deficient mice reach puberty. PMID:24095779

  8. Increased osteoblast function on PLGA composites containing nanophase titania.

    PubMed

    Webster, Thomas J; Smith, Tyler A

    2005-09-15

    Nanotechnology creates materials that potentially outperform, at several boundaries, existing materials in terms of mechanical, electrical, catalytic, and optical properties. However, despite their promise to mimic the surface roughness cells experience in vivo, the use of nanophase materials in biological applications remains to date largely unexplored. The objective of the present in vitro study was, therefore, to determine whether when added to a polymer scaffold, nanophase compared to conventional ceramics enhance functions of osteoblasts (or bone-forming cells). Results from this study provided the first evidence that functions (specifically, adhesion, synthesis of alkaline phosphatase, and deposition of calcium-containing mineral) of osteoblasts increased on poly-lactic-co-glycolic acid (PLGA) scaffolds containing nanophase compared to conventional grain size titania with greater weight percentage (from 10-30 wt %). Because the chemistry, material phase, porosity (%), and pore size of the composites were similar, this study implies that the surface features created by adding nanophase compared to conventional titania was a key parameter that enhanced functions of osteoblasts. In this manner, the study adds another novel property of nanophase ceramics: their ability to promote osteoblast functions in vitro when added to a polymer scaffold. For this reason, nanophase ceramics (and nanomaterials in general) deserve further attention as orthopedic tissue engineering materials.

  9. Local origins impart conserved bone type-related differences in human osteoblast behaviour.

    PubMed

    Shah, M; Gburcik, V; Reilly, P; Sankey, R A; Emery, R J; Clarkin, C E; Pitsillides, A A

    2015-03-04

    Osteogenic behaviour of osteoblasts from trabecular, cortical and subchondral bone were examined to determine any bone type-selective differences in samples from both osteoarthritic (OA) and osteoporotic (OP) patients. Cell growth, differentiation; alkaline phosphatase (TNAP) mRNA and activity, Runt-related transcription factor-2 (RUNX2), SP7-transcription factor (SP7), bone sialoprotein-II (BSP-II), osteocalcin/bone gamma-carboxyglutamate (BGLAP), osteoprotegerin (OPG, TNFRSF11B), receptor activator of nuclear factor-κβ ligand (RANKL, TNFSF11) mRNA levels and proangiogenic vascular endothelial growth factor-A (VEGF-A) mRNA and protein release were assessed in osteoblasts from paired humeral head samples from age-matched, human OA/OP (n = 5/4) patients. Initial outgrowth and increase in cell number were significantly faster (p < 0.01) in subchondral and cortical than trabecular osteoblasts, in OA and OP, and this bone type-related differences were conserved despite consistently faster growth in OA. RUNX2/SP7 levels and TNAP mRNA and protein activity were, however, greater in trabecular than subchondral and cortical osteoblasts in OA and OP. BSP-II levels were significantly greater in trabecular and lowest in cortical osteoblasts in both OA and OP. In contrast, BGLAP levels showed divergent bone type-selective behaviour; highest in osteoblasts from subchondral origins in OA and trabecular origins in OP. We found virtually identical bone type-related differences, however, in TNFRSF11B:TNFSF11 in OA and OP, consistent with greater potential for paracrine effects on osteoclasts in trabecular osteoblasts. Subchondral osteoblasts (OA) exhibited highest VEGF-A mRNA levels and release. Our data indicate that human osteoblasts in trabecular, subchondral and cortical bone have inherent, programmed diversity, with specific bone type-related differences in growth, differentiation and pro-angiogenic potential in vitro.

  10. OSU53 Rescues Human OB-6 Osteoblastic Cells from Dexamethasone through Activating AMPK Signaling.

    PubMed

    Xu, Dawei; Zhao, Wei; Zhu, Xinhui; Fan, Jianbo; Cui, Shengyu; Sun, Yuyu; Chen, Xiang; Liu, Wei; Cui, Zhi-Ming

    2016-01-01

    Excessive dexamethasone (Dex) application causes osteoblast cell death, which could lead to osteoporosis or osteonecrosis. AMP-activated protein kinase (AMPK) activation is shown to protect osteoblasts/osteoblastic cells from Dex. In this report, we tested the potential effect of OSU53, a novel AMPK activator, in Dex-treated osteoblastic cells. We show that OSU53 activated AMPK signaling in human OB-6 osteoblastic cells. Further, Dex-induced osteoblastic OB-6 cell death and apoptosis were largely attenuated with pre-treatment with OSU53. OSU53 was more efficient than other known AMPK activators (A-769662 and Compound 13) in protecting OB-6 cells against Dex. AMPK activation is required for OSU53-induced actions in OB-6 cells. AMPKα shRNA knockdown or dominant-negative mutation (dn-AMPKα T172A) almost completely blocked OSU53-induced AMPK activation and OB-6 cell protection against Dex. Further studies showed that OSU53 increased NADPH (nicotinamide adenine dinucleotide phosphate) activity and alleviated Dex-induced oxidative stress in OB-6 cells. Such effects by OSU53 were again almost abolished with AMPKα shRNA or dn-AMPKα in OB-6 cells. Together, these results demonstrate that OSU53 protects osteoblastic cells from Dex possibly via activating AMPK-dependent signaling. PMID:27632213

  11. OSU53 Rescues Human OB-6 Osteoblastic Cells from Dexamethasone through Activating AMPK Signaling

    PubMed Central

    Xu, Dawei; Zhao, Wei; Zhu, Xinhui; Fan, Jianbo; Cui, Shengyu; Sun, Yuyu; Chen, Xiang; Liu, Wei; Cui, Zhi-ming

    2016-01-01

    Excessive dexamethasone (Dex) application causes osteoblast cell death, which could lead to osteoporosis or osteonecrosis. AMP-activated protein kinase (AMPK) activation is shown to protect osteoblasts/osteoblastic cells from Dex. In this report, we tested the potential effect of OSU53, a novel AMPK activator, in Dex-treated osteoblastic cells. We show that OSU53 activated AMPK signaling in human OB-6 osteoblastic cells. Further, Dex-induced osteoblastic OB-6 cell death and apoptosis were largely attenuated with pre-treatment with OSU53. OSU53 was more efficient than other known AMPK activators (A-769662 and Compound 13) in protecting OB-6 cells against Dex. AMPK activation is required for OSU53-induced actions in OB-6 cells. AMPKα shRNA knockdown or dominant-negative mutation (dn-AMPKα T172A) almost completely blocked OSU53-induced AMPK activation and OB-6 cell protection against Dex. Further studies showed that OSU53 increased NADPH (nicotinamide adenine dinucleotide phosphate) activity and alleviated Dex-induced oxidative stress in OB-6 cells. Such effects by OSU53 were again almost abolished with AMPKα shRNA or dn-AMPKα in OB-6 cells. Together, these results demonstrate that OSU53 protects osteoblastic cells from Dex possibly via activating AMPK-dependent signaling. PMID:27632213

  12. Osteoblast adhesion to clodronate-hydroxyapatite composite

    NASA Astrophysics Data System (ADS)

    Luo, E.; Liu, X.; Wei, S. C.; Cai, X. X.; Hu, J.

    2008-11-01

    The present study combined clodronate, one of bisphosphonates, to the surface of HA scaffold by chelation. The cell culture test and gene expression test were performed and evaluated the effect of clodronate modifying the HA on co-culturing osteoblast with HA scaffold in vitro. Characteristic peaks in XPS and FT-IR spectra indicated clodronate being immobilized on the surface of HA. The cell culture test indicated that the cells actively proliferated on the scaffolds. The results of RT-PCR showed there was no significant difference between two groups in expression of RANKL gene, while the expression of OPG gene showed higher expression in Clodronate-HA group. Clodronate had no obvious effect on the cytocompatibility of HA, and Clodronate-HA might be used as bone scaffold with potential ability to improve osteogenesis.

  13. Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: Role of β-catenin/connexin43 association

    SciTech Connect

    Talhouk, Rabih S.; Fares, Mohamed-Bilal; Rahme, Gilbert J.; Hariri, Hanaa H.; Rayess, Tina; Dbouk, Hashem A.; Bazzoun, Dana; Al-Labban, Dania; El-Sabban, Marwan E.

    2013-12-10

    Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of β-catenin, α-catenin and ZO-2, it decreased nuclear levels of β-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, β-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, β-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering β-catenin away from nucleus. - Highlights: • Cx43 over-expressing MCF-7 and MDA-MB-231 were grown in 2D and 3D cultures. • Proliferation and growth morphology were affected in a context dependent manner. • Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced. • Cx43-mediated gap junction complex assembly correlated with observed changes. • We propose that membranous Cx43 sequesters β-catenin away from the nucleus.

  14. Strontium ranelate increases osteoblast activity.

    PubMed

    Almeida, Monica Marletti; Nani, Edson Parra; Teixeira, Lucas Novaes; Peruzzo, Daiane Cristina; Joly, Júlio César; Napimoga, Marcelo Henrique; Martinez, Elizabeth Ferreira

    2016-06-01

    Strontium ranelate (SR) is the first generation of a new class of medication for osteoporosis, which is capable of inducing bone formation and, to a certain extent, inhibiting bone resorption. The aim of this study was to evaluate the in vitro effects of SR on osteoblastic cell cultures. MC3TE-E1 cells were seeded in 24-well plates at a density of 2×10(4) cells/well and exposed to SR at 0.05, 0.1, and 0.5mM. The following parameters were assayed: 1) Cell proliferation by hemocytometer counting after 24, 48 and 72h, 2) Cell viability by MTT assay after 24, 48 and 72h, 3) Type I Collagen and Osteopontin (OPN) quantification by Western Blotting, ELISA, and Real Time PCR after 48h, 3) Immunolocalization of fibronectin (FN) by epifluorescence, and 4) matrix mineralization by Alizarin Red staining after 14days. After 24, 48 and 72h, the cell proliferation and viability were not affected by SR at 0.05 and 0.1mM (p>0.05). However, cell cultures exposed to SR at 0.5mM exhibited a decrease in both cell proliferation and cell viability in all time points assayed (p<0.05). High levels of protein and mRNA for Type I Collagen and OPN were detected in cultures exposed to SR, particularly at 0.5mM (p<0.05). SR allowed the expression of FN in osteoblastic cell cultures as observed by epifluorescence analysis. The mineralized bone-like nodule formation was affected in a concentration-dependent manner by SR, with large bone-like nodules being detected in osteoblastic cell cultures exposed to SR at 0.5mM. In conclusion, these results suggest that SR can accelerate acquisition of the osteoblastic phenotype, which explains, at least in part, the rebalancing of bone turnover in favor of bone formation. PMID:27157549

  15. Developing osteoblasts as an endpoint for the mouse embryonic stem cell test.

    PubMed

    Chen, Xinrong; Hansen, Deborah K; Merry, Gwenn; DeJarnette, Christian; Nolen, Greg; Sloper, Daniel; Fisher, J Edward; Harrouk, Wafa; Tassinari, Melissa S; Inselman, Amy L

    2015-06-01

    The mouse Embryonic Stem cell Test (EST) using cardiomyocyte differentiation is a promising in vitro assay for detecting potential embryotoxicity; however, the addition of another differentiation endpoint, such as osteoblasts, may improve the predictive value of the test. A number of variables such as culture conditions and starting cell number were investigated. A 14 day direct plating method of D3 mouse embryonic stem cells (mESCs) was used to test the predictivity of osteoblast differentiation as an endpoint in the EST. Twelve compounds were tested using the prediction model developed in the ECVAM validation study. Eight of the compounds selected from the EST validation study served as model compounds; four additional compounds known to produce skeletal defects were also tested. Our results indicate comparable chemical classification between the validated cardiomyocyte endpoint and the osteoblast endpoint. These results suggest that differentiation to osteoblasts may provide confirmatory information in predicting embryotoxicity.

  16. Osteoblast Menin Regulates Bone Mass in Vivo*

    PubMed Central

    Kanazawa, Ippei; Canaff, Lucie; Abi Rafeh, Jad; Angrula, Aarti; Li, Jingjing; Riddle, Ryan C.; Boraschi-Diaz, Iris; Komarova, Svetlana V.; Clemens, Thomas L.; Murshed, Monzur; Hendy, Geoffrey N.

    2015-01-01

    Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-β (TGF-β) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-β, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1f/f) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1f/f mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance. PMID:25538250

  17. Glycosaminoglycans enhance osteoblast differentiation of bone marrow derived human mesenchymal stem cells.

    PubMed

    Mathews, Smitha; Mathew, Suja Ann; Gupta, Pawan Kumar; Bhonde, Ramesh; Totey, Satish

    2014-02-01

    Extracellular matrix plays an important role in regulating cell growth and differentiation. The biomimetic approach of cell-based tissue engineering is based on mirroring this in vivo micro environment for developing a functional tissue engineered construct. In this study, we treated normal tissue culture plates with selected extracellular matrix components consisting of glycosaminoglycans such as chondroitin-4-sulphate, dermatan sulphate, chondroitin-6-sulphate, heparin and hyaluronic acid. Mesenchymal stem cells isolated from adult human bone marrow were cultured on the glycosaminoglycan treated culture plates to evaluate their regulatory role in cell growth and osteoblast differentiation. Although no significant improvement on human mesenchymal stem cell adhesion and proliferation was observed on the glycosaminoglycan-treated tissue culture plates, there was selective osteoblast differentiation, indicating its potential role in differentiation rather than proliferation. Osteoblast differentiation studies showed high osteogenic potential for all tested glycosaminoglycans except chondroitin-4-sulphate. Osteoblast differentiation-associated genes such as osterix, osteocalcin, integrin binding sialoprotein, osteonectin and collagen, type 1, alpha 1 showed significant upregulation. We identified osterix as the key transcription factor responsible for the enhanced bone matrix deposition observed on hyaluronic acid, heparin and chondroitin-6-sulphate. Hyaluronic acid provided the most favourable condition for osteoblast differentiation and bone matrix synthesis. Our results confirm and emphasise the significant role of extracellular matrix in regulating cell differentiation. To summarise, glycosaminoglycans of extracellular matrix played a significant role in regulating osteoblast differentiation and could be exploited in the biomimetic approach of fabricating or functionalizing scaffolds for stem cell based bone tissue engineering.

  18. Are cementoblasts a subpopulation of osteoblasts or a unique phenotype?

    PubMed

    Bosshardt, D D

    2005-05-01

    Experimental studies have shown a great potential for periodontal regeneration. The limitations of periodontal regeneration largely depend on the regenerative potential at the root surface. Cellular intrinsic fiber cementum (CIFC), so-called bone-like tissue, may form instead of the desired acellular extrinsic fiber cementum (AEFC), and the interfacial tissue bonding may be weak. The periodontal ligament harbors progenitor cells that can differentiate into periodontal ligament fibroblasts, osteoblasts, and cementoblasts, but their precise location is unknown. It is also not known whether osteoblasts and cementoblasts arise from a common precursor cell line, or whether distinct precursor cell lines exist. Thus, there is limited knowledge about how cell diversity evolves in the space between the developing root and the alveolar bone. This review supports the hypothesis that AEFC is a unique tissue, while CIFC and bone share some similarities. Morphologically, functionally, and biochemically, however, CIFC is distinctly different from any bone type. There are several lines of evidence to propose that cementoblasts that produce both AEFC and CIFC are unique phenotypes that are unrelated to osteoblasts. Cementum attachment protein appears to be cementum-specific, and the expression of two proteoglycans, fibromodulin and lumican, appears to be stronger in CIFC than in bone. A theory is presented that may help explain how cell diversity evolves in the periodontal ligament. It proposes that Hertwig's epithelial root sheath and cells derived from it play an essential role in the development and maintenance of the periodontium. The role of enamel matrix proteins in cementoblast and osteoblast differentiation and their potential use for tissue engineering are discussed. PMID:15840773

  19. WISP-1 is an osteoblastic regulator expressed during skeletal development and fracture repair.

    PubMed

    French, Dorothy M; Kaul, Raji J; D'Souza, Aloma L; Crowley, Craig W; Bao, Min; Frantz, Gretchen D; Filvaroff, Ellen H; Desnoyers, Luc

    2004-09-01

    Wnt-1-induced secreted protein 1 (WISP-1) is a member of the CCN (connective tissue growth factor, Cyr61, NOV) family of growth factors. Experimental evidence suggests that CCN family members are involved in skeletogenesis and bone healing. To investigate the role of WISP-1 in osteogenic processes, we characterized its tissue and cellular expression and evaluated its activity in osteoblastic and chondrocytic cell culture models. During embryonic development, WISP-1 expression was restricted to osteoblasts and to osteoblastic progenitor cells of the perichondral mesenchyme. In vitro, we showed that WISP-1 expression in differentiating osteoblasts promotes BMP-2-induced osteoblastic differentiation. Using in situ and cell binding analysis, we demonstrated WISP-1 interaction with perichondral mesenchyme and undifferentiated chondrocytes. We evaluated the effect of WISP-1 on chondrocytes by generating stably transfected mouse chondrocytic cell lines. In these cells, WISP-1 increased proliferation and saturation density but repressed chondrocytic differentiation. Because of the similarity between skeletogenesis and bone healing, we also analyzed WISP-1 spatiotemporal expression in a fracture repair model. We found that WISP-1 expression recapitulates the pattern observed during skeletal development. Our data demonstrate that WISP-1 is an osteogenic potentiating factor promoting mesenchymal cell proliferation and osteoblastic differentiation while repressing chondrocytic differentiation. Therefore, we propose that WISP-1 plays an important regulatory role during bone development and fracture repair.

  20. Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2.

    PubMed

    Wang, Yu-Hsiung; Nemati, Reza; Anstadt, Emily; Liu, Yaling; Son, Young; Zhu, Qiang; Yao, Xudong; Clark, Robert B; Rowe, David W; Nichols, Frank C

    2015-12-01

    Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential

  1. Effects of Hypogravity on Osteoblast Differentiation

    NASA Technical Reports Server (NTRS)

    Globus, Ruth; Doty, Steven

    1997-01-01

    Weightbearing is essential for normal skeletal function. Without weightbearing, the rate of bone formation by osteoblasts decreases in the growing rat. Defective formation may account for the decrease in the maturation, strength and mass of bone that is caused by spaceflight. These skeletal defects may be mediated by a combination of physiologic changes triggered by spaceflight, including skeletal unloading, fluid shifts, and stress-induced endocrine factors. The fundamental question of whether the defects in osteoblast function due to weightlessness are mediated by localized skeletal unloading or by systemic physiologic adaptations such as fluid shifts has not been answered. Furthermore, bone-forming activity of osteoblasts during unloading may be affected by paracrine signals from vascular, monocytic, and neural cells that also reside in skeletal tissue. Therefore we proposed to examine whether exposure of cultured rat osteoblasts to spaceflight inhibits cellular differentiation and impairs mineralization when isolated from the influence of both systemic factors and other skeletal cells.

  2. Mechanoresponses of human primary osteoblasts grown on carbon nanotubes.

    PubMed

    Kroustalli, A; Kotsikoris, V; Karamitri, A; Topouzis, S; Deligianni, D

    2015-03-01

    Bone mechanotransduction is strongly influenced by the biomaterial properties. A good understanding of these mechanosensory mechanisms in bone has the potential to provide new strategies in the highly evolving field of bone tissue engineering. The aim of the present investigation was to study the interactive effects of local mechanical stimuli on multiwalled carbon nanotubes (MWCNTs)/osteoblast interface, using an in vitro model that allows the study of cell growth, attachment and differentiation. Strain was applied at physiological levels [strain magnitudes 500 microstrain (μɛ), at frequency of load application 0.5 Hz]. The effect of mechanical strain and substrate was thus studied by measuring the messenger RNA expression of alkaline phosphatase, vinculin, collagen 1A, and integrins β1, β3, α4, and αv, using real-time quantitative polymerase chain reaction. The osteoblasts grown on MWCNTs displayed quick adaptation to the new environment by modulating the expression of key adhesion integrins. Furthermore, the addition of mechanical strain interplayed with the extracellular matrix and was efficiently transduced by cells grown on MWCNTs, providing stronger adhesion and survival. MWCNTs are therefore a material perfectly compatible with osteoblast differentiation, adhesion, and growth, and should be further evaluated, to derive new-generation biomaterial scaffolds for the treatment of skeletal defects which require bone reconstruction.

  3. Microcracks induce osteoblast alignment and maturation on hydroxyapatite scaffolds

    NASA Astrophysics Data System (ADS)

    Shu, Yutian

    Physiological bone tissue is a mineral/collagen composite with a hierarchical structure. The features in bone, such as mineral crystals, fibers, and pores can range from the nanometer to the centimeter in size. Currently available bone tissue scaffolds primarily address the chemical composition, pore size, and pore size distribution. While these design parameters are extensively investigated for mimicking bone function and inducing bone regeneration, little is known about microcracks, which is a prevalent feature found in fractured bone in vivo and associated with fracture healing and repair. Since the purpose of bone tissue engineering scaffold is to enhance bone regeneration, the coincidence of microcracks and bone densification should not be neglected but rather be considered as a potential parameter in bone tissue engineering scaffold design. The purpose of this study is to test the hypothesis that microcracks enhance bone healing. In vitro studies were designed to investigate the osteoblast (bone forming cells) response to microcracks in dense (94%) hydroxyapatite substrates. Microcracks were introduced using a well-established Vickers indentation technique. The results of our study showed that microcracks induced osteoblast alignment, enhanced osteoblast attachment and more rapid maturation. These findings may provide insight into fracture healing mechanism(s) as well as improve the design of bone tissue engineering orthopedic scaffolds for more rapid bone regeneration.

  4. Acidosis inhibits mineralization in human osteoblasts.

    PubMed

    Takeuchi, Shoko; Hirukawa, Koji; Togari, Akifumi

    2013-09-01

    Osteoblasts and osteoclasts maintain bone volume. Acidosis affects the function of these cells including mineral metabolism. We examined the effect of acidosis on the expression of transcription factors and mineralization in human osteoblasts in vitro. Human osteoblasts (SaM-1 cells) derived from the ulnar periosteum were cultured with α-MEM containing 50 μg/ml ascorbic acid and 5 mM β-glycerophosphate (calcifying medium). Acidosis was induced by incubating the SaM-1 cells in 10 % CO₂ (pH approximately 7.0). Mineralization, which was augmented by the calcifying medium, was completely inhibited by acidosis. Acidosis depressed c-Jun mRNA and increased osteoprotegerin (OPG) production in a time-dependent manner. Depressing c-Jun mRNA expression using siRNA increased OPG production and inhibited mineralization. In addition, depressing OPG mRNA expression with siRNA enhanced mineralization in a dose-dependent manner. Acidosis or the OPG protein strongly inhibited mineralization in osteoblasts from neonatal mice. The present study was the first to demonstrate that acidosis inhibited mineralization, depressed c-Jun mRNA expression, and induced OPG production in human osteoblasts. These results suggest that OPG is involved in mineralization via c-Jun in human osteoblasts.

  5. Specific Biomimetic Hydroxyapatite Nanotopographies Enhance Osteoblastic Differentiation and Bone Graft Osteointegration

    PubMed Central

    Loiselle, Alayna E.; Wei, Lai; Faryad, Muhammad; Paul, Emmanuel M.; Lewis, Gregory S.; Gao, Jun; Lakhtakia, Akhlesh

    2013-01-01

    Impaired healing of cortical bone grafts represents a significant clinical problem. Cadaveric bone grafts undergo extensive chemical processing to decrease the risk of disease transmission; however, these processing techniques alter the bone surface and decrease the osteogenic potential of cells at the healing site. Extensive work has been done to optimize the surface of bone grafts, and hydroxyapatite (HAP) and nanotopography both increase osteoblastic differentiation. HAP is the main mineral component of bone and can enhance osteoblastic differentiation and bone implant healing in vivo, while nanotopography can enhance osteoblastic differentiation, adhesion, and proliferation. This is the first study to test the combined effects of HAP and nanotopographies on bone graft healing. With the goal of identifying the optimized surface features to improve bone graft healing, we tested the hypothesis that HAP-based nanotopographic resurfacing of bone grafts improves integration of cortical bone grafts by enhancing osteoblastic differentiation. Here we show that osteoblastic cells cultured on processed bones coated with specific-scale (50–60 nm) HAP nanotopographies display increased osteoblastic differentiation compared to cells on uncoated bone, bones coated with poly-l-lactic acid nanotopographies, or other HAP nanotopographies. Further, bone grafts coated with 50–60-nm HAP exhibited increased formation of new bone and improved healing, with mechanical properties equivalent to live autografts. These data indicate the potential for specific HAP nanotopographies to not only increase osteoblastic differentiation but also improve bone graft incorporation, which could significantly increase patient quality of life after traumatic bone injuries or resection of an osteosarcoma. PMID:23510012

  6. Does collagen trigger the recruitment of osteoblasts into vacated bone resorption lacunae during bone remodeling?

    PubMed

    Abdelgawad, Mohamed Essameldin; Søe, Kent; Andersen, Thomas Levin; Merrild, Ditte M H; Christiansen, Peer; Kjærsgaard-Andersen, Per; Delaisse, Jean-Marie

    2014-10-01

    Osteoblast recruitment during bone remodeling is obligatory to re-construct the bone resorbed by the osteoclast. This recruitment is believed to be triggered by osteoclast products and is therefore likely to start early during the remodeling cycle. Several osteoclast products with osteoblast recruitment potential are already known. Here we draw the attention on the osteoblast recruitment potential of the collagen that is freshly demineralized by the osteoclast. Our evidence is based on observations on adult human cancellous bone, combined with in vitro assays. First, freshly eroded surfaces where osteoblasts have to be recruited show the presence of non-degraded demineralized collagen and close cell-collagen interactions, as revealed by electron microscopy, while surface-bound collagen strongly attracts osteoblast lineage cells in a transmembrane migration assay. Compared with other extracellular matrix molecules, collagen's potency was superior and only equaled by fibronectin. Next, the majority of the newly recruited osteoblast lineage cells positioned immediately next to the osteoclasts exhibit uPARAP/Endo180, an endocytic collagen receptor reported to be involved in collagen internalization and cell migration in various cell types, and whose inactivation is reported to lead to lack of bone formation and skeletal deformities. In the present study, an antibody directed against this receptor inhibits collagen internalization in osteoblast lineage cells and decreases to some extent their migration to surface-bound collagen in the transmembrane migration assay. These complementary observations lead to a model where collagen demineralized by osteoclasts attracts surrounding osteoprogenitors onto eroded surfaces, and where the endocytic collagen receptor uPARAP/Endo180 contributes to this migration, probably together with other collagen receptors. This model fits recent knowledge on the position of osteoprogenitor cells immediately next to remodeling sites in adult

  7. MiR-214 regulates the function of osteoblast under simulated microgravity by targeting ATF4

    NASA Astrophysics Data System (ADS)

    Li, Yingxian; Wang, Xiaogang; Li, Qi; Lv, Ke; Wan, Yumin; Li, Yinghui; Bai, Yanqiang

    Background: MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3'-untranslated region (UTR) of mRNAs, resulting in translational repression. Growing evidence shows that microRNAs (miRNAs) regu-late various developmental and homeostatic events in vertebrates and invertebrates. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass; How-ever, the physiological role of non-coding small RNAs, especially miRNAs, in osteoblast dif-ferentiation remains elusive. Methods: To study the potential involvement of miRNAs in osteoblast differentiation under stimulated microgravity, we analyzed the expression of 20 bone relative miRNAs using real time PCR platform to find particularly miRNAs whose expression is altered during osteoblast differentiation. TargetScan, miRBase and Miranda were used to predict the target gene of candidate miRNA. To investigate whether ATF4 can be directly targeted by miR-214, we engineered luciferase reporters that have either the wild-type 3'UTRs of these genes, or the mutant UTRs with a 6 base pair (bp) deletion in the target sites. Lastly, to address the in vivo role of miR-214 in bone formation, tail suspension mice model was used to simulate the change of osteoblast function and bone loss. Results: Recent studies have sug-gested that miRNAs might play a role in osteoblast differentiation and bone formation. Here, we identify miR-214 in MC3T3-E1 cells, which is a primary mouse osteoblasts cell line, to promote osteoblast differentiation by repressing Activating Transcription Factor4 (ATF4) ex-pression at the posttranscriptional level. What is more, miR-214 was found to be transcribed in C2C12 cells during bone morphogenetic protein 2-induced (BMP2-induced) osteogenesis, and overexpression of miR-214 attenuated BMP2-induced osteoblastogenesis

  8. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    SciTech Connect

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha; Levi, Edi; Rishi, Arun K.; Datta, Nabanita S.

    2013-07-12

    suggesting that PTH utilized an Extracellular Signal Regulated Kinase (ERK)-independent but p38 dependent pathway to regulate CARP-1 protein expression in osteoblasts. Immunofluorescence staining of differentiated osteoblasts further revealed nuclear to cytoplasmic translocation of CARP-1 protein following PTH treatment. Collectively, our studies identified CARP-1 for the first time in osteoblasts and suggest its potential role in PTH signaling and bone anabolic action.

  9. Chronic exposure to hexachlorobenzene results in down-regulation of connexin43 in the breast.

    PubMed

    Delisle, Ariane; Ferraris, Emanuelle; Plante, Isabelle

    2015-11-01

    Decreased expression of connexins has been associated with cancer, but the underlying mechanisms are poorly understood. We have previously shown that a 5 day exposure to hexachlorobenzene (HCB) resulted in decreased connexins expression in hepatocytes 45 days later, and that this down-regulation was linked to activation of Akt through the ILK pathway. Because HCB promotes cancer in both the liver and breast, the present study aimed to determine if the mechanisms are similar in both tissues. MCF-12A breast cells were thus transfected with vectors coding for either Akt or a constitutively active form of Akt. In those cells, activation of Akt was correlated with decreased Cx43 levels. Female rats were then exposed to HCB by gavage either following the same protocol used previously for the liver or through a chronic exposure. While no changes were observed after the 5 days exposure protocol, chronic exposure to HCB resulted in increased Akt levels and decreased Cx43 levels in breast cells. In vitro, Akt was activated in MCF-12A cells exposed to HCB either for 7 days or chronically, but no changes were observed in junctional proteins. Together, these results suggested that, while activation of Akt can decrease Cx43 expression in breast cells in vitro, other mechanisms are involved during HCB exposure, leading to a decrease in Cx43 levels in a model- and duration-dependent manner. Finally, we showed that HCB effects are tissue specific, as we did not observe the same results in breast and liver tissues.

  10. Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43 in osteocytes

    PubMed Central

    Qin, An; Pavlos, Nathan J.; Wang, Tao; Song, Kai; Wang, Yan; Chen, Lianzhi; Zhou, Lin; Jiang, Qing; Takayanagi, Hiroshi; Yan, Sheng; Zheng, Minghao

    2016-01-01

    Osteocytes comprising over 90% of the bone cell population are highly susceptible to the adverse effects of glucocorticoids (GC) administration. Here we observed that Dexamethasone (Dex) induces a robust cytoskeleton rearrangement and decreases Cx43 protein expression in osteocyte-like MLO-Y4 cells. Using a Dmp1Cre-mT/mG osteocyte ex vivo culture system, we found significant shortening of dendritic processes in primary osteocytes following Dex administration. Loss of dendritic processes is a consequence of reduced Cx43 connectivity upon Dex induced autophagy in both RFP-GFP-LC3B transfected MLO-Y4 cells and primary calvarial osteocytes from LC3GFP transgenic mice. Upon the induction of autophagy by Dex, Cx43 was internalized into autophagosome/autolysosomes and degraded by autophagy. The degradation was attenuated following lysosomal inhibition using chloroquine (CLQ) and suppression of autophagy by Atg5 silencing. Inhibition Akt-mTORC1 signaling by Dex induces autophagy subsequently resulting in Cx43 degradation. Activation of Akt phosphorylation by IGF-1 attenuated Dex induced autophagy and degradation of Cx43. Together, we demonstrated that GC impair osteocyte cell-cell connectivity via autophagy mediated degradation of Cx43 through inhibition of the Akt-mTORC1 signaling. This may account for the deleterious effect of GC-induced bone loss. PMID:27127181

  11. Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43 in osteocytes.

    PubMed

    Gao, Junjie; Cheng, Tak Sum; Qin, An; Pavlos, Nathan J; Wang, Tao; Song, Kai; Wang, Yan; Chen, Lianzhi; Zhou, Lin; Jiang, Qing; Takayanagi, Hiroshi; Yan, Sheng; Zheng, Minghao

    2016-05-10

    Osteocytes comprising over 90% of the bone cell population are highly susceptible to the adverse effects of glucocorticoids (GC) administration. Here we observed that Dexamethasone (Dex) induces a robust cytoskeleton rearrangement and decreases Cx43 protein expression in osteocyte-like MLO-Y4 cells. Using a Dmp1Cre-mT/mG osteocyte ex vivo culture system, we found significant shortening of dendritic processes in primary osteocytes following Dex administration. Loss of dendritic processes is a consequence of reduced Cx43 connectivity upon Dex induced autophagy in both RFP-GFP-LC3B transfected MLO-Y4 cells and primary calvarial osteocytes from LC3GFP transgenic mice. Upon the induction of autophagy by Dex, Cx43 was internalized into autophagosome/autolysosomes and degraded by autophagy. The degradation was attenuated following lysosomal inhibition using chloroquine (CLQ) and suppression of autophagy by Atg5 silencing. Inhibition Akt-mTORC1 signaling by Dex induces autophagy subsequently resulting in Cx43 degradation.Activation of Akt phosphorylation by IGF-1 attenuated Dex induced autophagy and degradation of Cx43. Together, we demonstrated that GC impair osteocyte cell-cell connectivity via autophagy mediated degradation of Cx43 through inhibition of the Akt-mTORC1 signaling. This may account for the deleterious effect of GC-induced bone loss.

  12. Phenotypic transformation of smooth muscle cells from porcine coronary arteries is associated with connexin 43

    PubMed Central

    ZHANG, XUMIN; WANG, XIAODONG; ZHOU, XIAOHUI; MA, XIAOYE; YAO, YIAN; LIU, XUEBO

    2016-01-01

    The current study aimed to investigate the relevance of the gap junction protein connexin Cx43 in coronary artery smooth muscle cell (SMC) heterogeneity and coronary artery restenosis. SMCs were isolated from the coronary artery of 3-month-old pigs using enzymatic digestion. Two distinct SMC populations were isolated: Rhomboid (R) and spindle-shaped (S) cells. S-SMCs exhibited relatively lower rates of proliferation, exhibiting a classic ''hills-and valleys'' growth pattern; R-SMCs displayed increased proliferation rates, growing as mono- or multi-layers. Immunofluorescent staining, polymerase chain reaction and western blotting were used to assess the expression of Cx40 and Cx43 in SMCs. For further evaluation, cultured SMCs were treated with 10 ng/ml platelet-derived growth factor (PDGF)-BB with or without the gap junction blocker 18α-glycyrrhetinic acid. Stent-induced restenosis was assessed in vivo. Different expression patterns were observed for Cx40 and Cx43 in R- and S-SMCs. Cx40 was the most abundant Cx in S-SMCs, whereas CX43 was identified at relatively higher levels than Cx40 in R-SMCs. Notably, PDGF-BB converted S-SMCs to R-SMCs, with increased Cx43 expression, while 18α-glycyrrhetinic acid inhibited the PDGF-BB-induced phenotypic alterations in S-SMCs. Additionally, restenosis was confirmed in pigs 1-month subsequent to stent placement. R-SMCs were the major cell population isolated from stent-induced restenosis artery tissues, and exhibited markedly increased Cx43 expression, in accordance with the in vitro data described above. In conclusion, the phenotypic transformation of coronary artery SMCs is closely associated with Cx43, which is involved in restenosis. These observations provide a basis for the use of Cx43 as a novel target in restenosis prevention. PMID:27175888

  13. Reduced connexin 43 in eutopic endometrium and cultured endometrial stromal cells from subjects with endometriosis

    PubMed Central

    Yu, Jie; Boicea, Anisoara; Barrett, Kara L.; James, Christopher O.; Bagchi, Indrani C.; Bagchi, Milan K.; Nezhat, Ceana; Sidell, Neil; Taylor, Robert N.

    2014-01-01

    Accumulating evidence indicates that reduced fecundity associated with endometriosis reflects a failure of embryonic receptivity. Microdomains composed of endometrial gap junctions, which facilitate cell–cell communication, may be implicated. Pharmacological or genetic inhibition of connexin (Cx) 43 block human endometrial cell differentiation in vitro and conditional uterine deletion of Cx43 alleles cause implantation failure in mice. The aim of this study was to determine whether women with endometriosis have reduced eutopic endometrial Cx43. Cx26 acted as a control. Endometrial biopsies were collected from age, race and cycle phase-matched women without (15 controls) or with histologically confirmed endometriosis (15 cases). Immunohistochemistry confirmed a predominant localization of Cx43 in the endometrial stroma, whereas Cx26 was confined to the epithelium. Cx43 immunostaining was reduced in eutopic biopsies of endometriosis subjects and western blotting of tissue lysates confirmed lower Cx43 levels in endometriosis cases, with Cx43/β-actin ratios =3.4 ± 1.5 in control and =1.2 ± 0.3 in endometriosis biopsies (P < 0.01). When endometrial stromal cells (ESC) were isolated from endometriosis cases, Cx43 levels and scrape loading-dye transfer were reduced by ∼45% compared with ESC from controls. In vitro decidualization of ESC derived from endometriosis versus control subjects resulted in lesser epithelioid transformation and a significantly reduced up-regulation of Cx43 protein (1.2 ± 0.2- versus 1.7 ± 0.4-fold, P < 0.01). No changes in Cx26 were observed. While basal steady-state levels of Cx43 mRNA did not differ with respect to controls, ESC from endometriosis cases failed to manifest a response to hormone treatment in vitro. In summary, eutopic endometrial Cx43 concentrations in endometriosis cases were <50% those of controls in vivo and in vitro, functional gap junctions were reduced and hormone-induced Cx43 mRNA levels were blunted. PMID:24270393

  14. Cell line specific modulation of connexin43 expression after exposure to ionizing radiation.

    PubMed

    Banaz-Yaşar, Ferya; Tischka, Rabea; Iliakis, George; Winterhager, Elke; Gellhaus, Alexandra

    2005-01-01

    Gap junctional intercellular communication plays a significant role in mediating radiation-induced bystander effects. However, the level of Cx43 itself is influenced by ionizing radiation, which could modify the bystander effect. Here we have investigated several cell lines for the modulation of Cx43 expression 24 h after irradiation with 5 Gy X-rays. The mouse endothelial cell line bEnd3 revealed a significantly elevated level of Cx43 already 15 min after exposure to X-rays, whereas human hybrid endothelial cells (EA.hy926) exhibited a transient downregulation of Cx43 mRNA. No obvious changes in the communication properties of the different cell lines could be observed after irradiation. The communication-deficient malignant human trophoblast cell line Jeg3 stably transfected with Cx43 did not reveal any induction of endogenous nor alteration in the exogenous Cx43 transcript level upon exposure to 5 Gy. Taken together, our data show a cell line specific modulation of Cx43 expression after exposure to X-rays.

  15. P6 Electroacupuncture Improved QTc Interval Prolongation by Upregulation of Connexin43 in Droperidol Treated Rats

    PubMed Central

    Zhao, Feng; Cui, Suyang; Huang, Libing

    2014-01-01

    Aim. This study investigated the effect of P6 EA on droperidol-induced QTc interval prolongation and Cx43 expression in ventricular muscle of rats. Methods. Twenty-four rats were randomly divided into control group (C), droperidol group (D), or EA group (E). C group rats were injected with normal saline. D group rats were injected with droperidol 0.13 mg/kg. E group rats were pretreated with EA at left P6 acupoint for 30 min and then injected with droperidol (0.13 mg/kg). QTc intervals were recorded at lead II in ECG within 120 min. Cx43 expression was measured by RT-PCR and western blotting. Result. Droperidol significantly prolonged QTc intervals compared with controls at 5 min, 10 min, 15 min, and 30 min (P < 0.05). P6 EA could significantly abbreviate the prolongation of QTc interval compared with droperidol group at 5 min, 10 min, 15 min, and 30 min (P < 0.05). Cx43 mRNA and proteins were significantly increased by P6 EA compared with droperidol group at 120 min (P < 0.05). There were no significant differences in Cx43 mRNA and protein expression between droperidol and control group at 120 min (P > 0.05). Conclusion. P6 EA could improve QTc interval prolongation induced by droperidol, which may relate to upregulation of Cx43 mRNA and protein. Antiemetic dose of droperidol had minor effects on Cx43 mRNA and protein expression at 120 min. PMID:25371698

  16. The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes

    PubMed Central

    Abudara, Verónica; Bechberger, John; Freitas-Andrade, Moises; De Bock, Marijke; Wang, Nan; Bultynck, Geert; Naus, Christian C.; Leybaert, Luc; Giaume, Christian

    2014-01-01

    In the brain, astrocytes represent the cellular population that expresses the highest amount of connexins (Cxs). This family of membrane proteins is the molecular constituent of gap junction channels and hemichannels that provide pathways for direct cytoplasm-to-cytoplasm and inside-out exchange, respectively. Both types of Cx channels are permeable to ions and small signaling molecules allowing astrocytes to establish dynamic interactions with neurons. So far, most pharmacological approaches currently available do not distinguish between these two channel functions, stressing the need to develop new specific molecular tools. In astrocytes two major Cxs are expressed, Cx43 and Cx30, and there is now evidence indicating that at least Cx43 operates as a gap junction channel as well as a hemichannel in these cells. Based on studies in primary cultures as well as in acute hippocampal slices, we report here that Gap19, a nonapeptide derived from the cytoplasmic loop of Cx43, inhibits astroglial Cx43 hemichannels in a dose-dependent manner, without affecting gap junction channels. This peptide, which not only selectively inhibits hemichannels but is also specific for Cx43, can be delivered in vivo in mice as TAT-Gap19, and displays penetration into the brain parenchyma. As a result, Gap19 combined with other tools opens up new avenues to decipher the role of Cx43 hemichannels in interactions between astrocytes and neurons in physiological as well as pathological situations. PMID:25374505

  17. Cell line specific modulation of connexin43 expression after exposure to ionizing radiation.

    PubMed

    Banaz-Yaşar, Ferya; Tischka, Rabea; Iliakis, George; Winterhager, Elke; Gellhaus, Alexandra

    2005-01-01

    Gap junctional intercellular communication plays a significant role in mediating radiation-induced bystander effects. However, the level of Cx43 itself is influenced by ionizing radiation, which could modify the bystander effect. Here we have investigated several cell lines for the modulation of Cx43 expression 24 h after irradiation with 5 Gy X-rays. The mouse endothelial cell line bEnd3 revealed a significantly elevated level of Cx43 already 15 min after exposure to X-rays, whereas human hybrid endothelial cells (EA.hy926) exhibited a transient downregulation of Cx43 mRNA. No obvious changes in the communication properties of the different cell lines could be observed after irradiation. The communication-deficient malignant human trophoblast cell line Jeg3 stably transfected with Cx43 did not reveal any induction of endogenous nor alteration in the exogenous Cx43 transcript level upon exposure to 5 Gy. Taken together, our data show a cell line specific modulation of Cx43 expression after exposure to X-rays. PMID:16531320

  18. Mutations in GJA1 (connexin 43) are associated with non-syndromic autosomal recessive deafness.

    PubMed

    Liu, X Z; Xia, X J; Adams, J; Chen, Z Y; Welch, K O; Tekin, M; Ouyang, X M; Kristiansen, A; Pandya, A; Balkany, T; Arnos, K S; Nance, W E

    2001-12-01

    Mutations in four members of the connexin gene family have been shown to underlie distinct genetic forms of deafness, including GJB2 [connexin 26 (Cx26)], GJB3 (Cx31), GJB6 (Cx30) and GJB1 (Cx32). We have found that alterations in a fifth member of this family, GJA1 (Cx43), appear to cause a common form of deafness in African Americans. We identified two different GJA1 mutations in four of 26 African American probands. Three were homozygous for a Leu-->Phe substitution in the absolutely conserved codon 11, whereas the other was homozygous for a Val-->Ala transversion at the highly conserved codon 24. Neither mutation was detected in DNA from 100 control subjects without deafness. Cx43 is expressed in the cochlea, as is demonstrated by PCR amplification from human fetal cochlear cDNA and by RT-PCR of mouse cochlear tissues. Immunohistochemical staining of mouse cochlear preparations showed immunostaining for Cx43 in non-sensory epithelial cells and in fibrocytes of the spiral ligament and the spiral limbus. To our knowledge this is the first alpha connexin gene to be associated with non-syndromic deafness. Cx43 must also play a critical role in the physiology of hearing, presumably by participating in the recycling of potassium to the cochlear endolymph. PMID:11741837

  19. Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cβ.

    PubMed

    Huang, Yu-Fang; Liao, Chih-Kai; Lin, Jau-Chen; Jow, Guey-Mei; Wang, Hwai-Shi; Wu, Jiahn-Chun

    2013-03-01

    Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of protein kinase C (PKC)α/βII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca(2+) chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCβ inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCβ signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS. PMID:23403203

  20. Domain-Specific Partitioning of Uterine Artery Endothelial Connexin43 and Caveolin-1.

    PubMed

    Ampey, Bryan C; Morschauser, Timothy J; Ramadoss, Jayanth; Magness, Ronald R

    2016-10-01

    Uterine vascular adaptations facilitate rises in uterine blood flow during pregnancy, which are associated with gap junction connexin (Cx) proteins and endothelial nitric oxide synthase. In uterine artery endothelial cells (UAECs), ATP activates endothelial nitric oxide synthase in a pregnancy (P)-specific manner that is dependent on Cx43 function. Caveolar subcellular domain partitioning plays key roles in ATP-induced endothelial nitric oxide synthase activation and nitric oxide production. Little is known regarding the partitioning of Cx proteins to caveolar domains or their dynamics with ATP treatment. We observed that Cx43-mediated gap junction function with ATP stimulation is associated with Cx43 repartitioning between the noncaveolar and caveolar domains. Compared with UAECs from nonpregnant (NP) ewes, levels of ATP, PGI2, cAMP, NOx, and cGMP were 2-fold higher (P<0.05) in pregnant UAECs. In pregnant UAECs, ATP increased Lucifer yellow dye transfer, a response abrogated by Gap27, but not Gap 26, indicating involvement of Cx43, but not Cx37. Confocal microscopy revealed domain partitioning of Cx43 and caveolin-1. In pregnant UAECs, LC/MS/MS analysis revealed only Cx43 in the caveolar domain. In contrast, Cx37 was located only in the noncaveolar pool. Western analysis revealed that ATP increased Cx43 distribution (1.7-fold; P=0.013) to the caveolar domain, but had no effect on Cx37. These data demonstrate rapid ATP-stimulated repartitioning of Cx43 to the caveolae, where endothelial nitric oxide synthase resides and plays an important role in nitric oxide-mediated increasing uterine blood flow during pregnancy. PMID:27572151

  1. Domain-Specific Partitioning of Uterine Artery Endothelial Connexin43 and Caveolin-1.

    PubMed

    Ampey, Bryan C; Morschauser, Timothy J; Ramadoss, Jayanth; Magness, Ronald R

    2016-10-01

    Uterine vascular adaptations facilitate rises in uterine blood flow during pregnancy, which are associated with gap junction connexin (Cx) proteins and endothelial nitric oxide synthase. In uterine artery endothelial cells (UAECs), ATP activates endothelial nitric oxide synthase in a pregnancy (P)-specific manner that is dependent on Cx43 function. Caveolar subcellular domain partitioning plays key roles in ATP-induced endothelial nitric oxide synthase activation and nitric oxide production. Little is known regarding the partitioning of Cx proteins to caveolar domains or their dynamics with ATP treatment. We observed that Cx43-mediated gap junction function with ATP stimulation is associated with Cx43 repartitioning between the noncaveolar and caveolar domains. Compared with UAECs from nonpregnant (NP) ewes, levels of ATP, PGI2, cAMP, NOx, and cGMP were 2-fold higher (P<0.05) in pregnant UAECs. In pregnant UAECs, ATP increased Lucifer yellow dye transfer, a response abrogated by Gap27, but not Gap 26, indicating involvement of Cx43, but not Cx37. Confocal microscopy revealed domain partitioning of Cx43 and caveolin-1. In pregnant UAECs, LC/MS/MS analysis revealed only Cx43 in the caveolar domain. In contrast, Cx37 was located only in the noncaveolar pool. Western analysis revealed that ATP increased Cx43 distribution (1.7-fold; P=0.013) to the caveolar domain, but had no effect on Cx37. These data demonstrate rapid ATP-stimulated repartitioning of Cx43 to the caveolae, where endothelial nitric oxide synthase resides and plays an important role in nitric oxide-mediated increasing uterine blood flow during pregnancy.

  2. Connexin45 interacts with zonula occludens-1 in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Laing, J. G.; Manley-Markowski, R. N.; Koval, M.; Civitelli, R.; Steinberg, T. H.

    2001-01-01

    Connexin43 (Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.

  3. Enhanced functions of osteoblasts on nanophase ceramics.

    PubMed

    Webster, T J; Ergun, C; Doremus, R H; Siegel, R W; Bizios, R

    2000-09-01

    Select functions of osteoblasts (bone-forming cells) on nanophase (materials with grain sizes less than 100 nm) alumina, titania, and hydroxyapatite (HA) were investigated using in vitro cellular models. Compared to conventional ceramics, surface occupancy of osteoblast colonies was significantly less on all nanophase ceramics tested in the present study after 4 and 6 days of culture. Osteoblast proliferation was significantly greater on nanophase alumina, titania, and HA than on conventional formulations of the same ceramic after 3 and 5 days. More importantly, compared to conventional ceramics, synthesis of alkaline phosphatase and deposition of calcium-containing mineral was significantly greater by osteoblasts cultured on nanophase than on conventional ceramics after 21 and 28 days. The results of the present study provided the first evidence of enhanced long-term (on the order of days to weeks) functions of osteoblasts cultured on nanophase ceramics; in this manner, nanophase ceramics clearly represent a unique and promising class of orthopaedic/dental implant formulations with improved osseointegrative properties.

  4. Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

    SciTech Connect

    Hong, Dun; Chen, Hai-Xiao; Yu, Hai-Qiang; Liang, Yong; Wang, Carrie; Lian, Qing-Quan; Deng, Hai-Teng; Ge, Ren-Shan

    2010-08-15

    Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.

  5. Skeletal Collagen Turnover by the Osteoblast

    NASA Technical Reports Server (NTRS)

    Partridge, Nicola C.

    1997-01-01

    Among the most overt negative changes experienced by man and experimental animals under conditions of weightlessness are the loss of skeletal mass and attendant hypercalciuria. These clearly result from some disruption in the balance between bone formation and bone resorption (i.e. remodelling) which appears to be due to a decrease in the functions of the osteoblast. In the studies funded by this project, the clonal osteoblastic cell line, UMR 106-01, has been used to investigate the regulation of collagenase and Tissue Inhibitors of MetalloProteases (TIMPs). This project has shed light on the comprehensive role of the osteoblast in the remodelling process, and, in so doing, provided some insight into how the process might be disrupted under conditions of microgravity.

  6. A 3D printed nano bone matrix for characterization of breast cancer cell and osteoblast interactions

    NASA Astrophysics Data System (ADS)

    Zhu, Wei; Castro, Nathan J.; Cui, Haitao; Zhou, Xuan; Boualam, Benchaa; McGrane, Robert; Glazer, Robert I.; Zhang, Lijie Grace

    2016-08-01

    Bone metastasis is one of the most prevalent complications of late-stage breast cancer, in which the native bone matrix components, including osteoblasts, are intimately involved in tumor progression. The development of a successful in vitro model would greatly facilitate understanding the underlying mechanism of breast cancer bone invasion as well as provide a tool for effective discovery of novel therapeutic strategies. In the current study, we fabricated a series of in vitro bone matrices composed of a polyethylene glycol hydrogel and nanocrystalline hydroxyapatite of varying concentrations to mimic the native bone microenvironment for the investigation of breast cancer bone metastasis. A stereolithography-based three-dimensional (3D) printer was used to fabricate the bone matrices with precisely controlled architecture. The interaction between breast cancer cells and osteoblasts was investigated in the optimized bone matrix. Using a Transwell® system to separate the two cell lines, breast cancer cells inhibited osteoblast proliferation, while osteoblasts stimulated breast cancer cell growth, whereas, both cell lines increased IL-8 secretion. Breast cancer cells co-cultured with osteoblasts within the 3D bone matrix formed multi-cellular spheroids in comparison to two-dimensional monolayers. These findings validate the use of our 3D printed bone matrices as an in vitro metastasis model, and highlights their potential for investigating breast cancer bone metastasis.

  7. Effects of fluoridation of porcine hydroxyapatite on osteoblastic activity of human MG63 cells

    NASA Astrophysics Data System (ADS)

    Li, Zhipeng; Huang, Baoxin; Mai, Sui; Wu, Xiayi; Zhang, Hanqing; Qiao, Wei; Luo, Xin; Chen, Zhuofan

    2015-06-01

    Biological hydroxyapatite, derived from animal bones, is the most widely used bone substitute in orthopedic and dental treatments. Fluorine is the trace element involved in bone remodeling and has been confirmed to promote osteogenesis when administered at the appropriate dose. To take advantage of this knowledge, fluorinated porcine hydroxyapatite (FPHA) incorporating increasing levels of fluoride was derived from cancellous porcine bone through straightforward chemical and thermal treatments. Physiochemical characteristics, including crystalline phases, functional groups and dissolution behavior, were investigated on this novel FPHA. Human osteoblast-like MG63 cells were cultured on the FPHA to examine cell attachment, cytoskeleton, proliferation and osteoblastic differentiation for in vitro cellular evaluation. Results suggest that fluoride ions released from the FPHA play a significant role in stimulating osteoblastic activity in vitro, and appropriate level of fluoridation (1.5 to 3.1 atomic percents of fluorine) for the FPHA could be selected with high potential for use as a bone substitute.

  8. Staphylococcus epidermidis serine--aspartate repeat protein G (SdrG) binds to osteoblast integrin alpha V beta 3.

    PubMed

    Claro, T; Kavanagh, N; Foster, T J; O'Brien, F J; Kerrigan, S W

    2015-06-01

    Staphylococcus epidermidis is the leading etiologic agent of orthopaedic implant infection. Contamination of the implanted device during insertion allows bacteria gain entry into the sterile bone environment leading to condition known as osteomyelitis. Osteomyelitis is characterised by weakened bones associated with progressive bone loss. The mechanism through which S. epidermidis interacts with bone cells to cause osteomyelitis is poorly understood. We demonstrate here that S. epidermidis can bind to osteoblasts in the absence of matrix proteins. S. epidermidis strains lacking the cell wall protein SdrG had a significantly reduced ability to bind to osteoblasts. Consistent with this, expression of SdrG in Lactococcus lactis resulted in significantly increased binding to the osteoblasts. Protein analysis identified that SdrG contains a potential integrin recognition motif. αVβ3 is a major integrin expressed on osteoblasts and typically recognises RGD motifs in its ligands. Our results demonstrate that S. epidermidis binds to recombinant purified αVβ3, and that a mutant lacking SdrG failed to bind. Blocking αVβ3 on osteoblasts significantly reduced binding to S. epidermidis. These studies are the first to identify a mechanism through which S. epidermidis binds to osteoblasts and potentially offers a mechanism through which implant infection caused by S. epidermidis leads to osteomyelitis.

  9. Porous hydroxyapatite and biphasic calcium phosphate ceramics promote ectopic osteoblast differentiation from mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Zhang, Lingli; Hanagata, Nobutaka; Maeda, Megumi; Minowa, Takashi; Ikoma, Toshiyuki; Fan, Hongsong; Zhang, Xingdong

    2009-04-01

    Because calcium phosphate (Ca-P) ceramics have been used as bone substitutes, it is necessary to investigate what effects the ceramics have on osteoblast maturation. We prepared three types of Ca-P ceramics with different Ca-P ratios, i.e. hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP), and biphasic calcium phosphate (BCP) ceramics with dense-smooth and porous structures. Comprehensive gene expression microarray analysis of mouse osteoblast-like cells cultured on these ceramics revealed that porous Ca-P ceramics considerably affected the gene expression profiles, having a higher potential for osteoblast maturation. In the in vivo study that followed, porous Ca-P ceramics were implanted into rat skeletal muscle. Sixteen weeks after the implantation, more alkaline-phosphatase-positive cells were observed in the pores of hydroxyapatite and BCP, and the expression of the osteocalcin gene (an osteoblast-specific marker) in tissue grown in pores was also higher in hydroxyapatite and BCP than in β-TCP. In the pores of any Ca-P ceramics, 16 weeks after the implantation, we detected the expressions of marker genes of the early differentiation stage of chondrocytes and the complete differentiation stage of adipocytes, which originate from mesenchymal stem cells, as well as osteoblasts. These marker gene expressions were not observed in the muscle tissue surrounding the implanted Ca-P ceramics. These observations indicate that porous hydroxyapatite and BCP had a greater potential for promoting the differentiation of mesenchymal stem cells into osteoblasts than β-TCP.

  10. Bisphosphonate stimulation of osteoblasts and osteoblastic metastasis as a mechanism of hypocalcaemia.

    PubMed

    Ho, Joon Wee

    2012-03-01

    Bisphosphonates are used in the oncological setting to treat and prevent skeletal-related events and preserve bone mineral density. Bisphosphonates also possess a hypocalcaemic effect. When undesired, hypocalcaemia can result in increased morbidity and complications. The currently understood mechanism of bisphosphonate-induced hypocalcaemia is by osteoclast inhibition. The effect of bisphosphonates on osteoblasts is less well understood. Laboratory studies demonstrate that bisphosphonates increase osteoblast and osteoblastic metastases maturation, activity and bone mineralization. We hypothesize that where large populations of osteoblasts exist increased mineralization may result in hypocalcaemia. Consequently patients with bone-metastatic prostate cancer may be more susceptible to symptomatic hypocalcaemia following bisphosphonate therapy. We are currently designing a study to investigate our hypothesis and to identify the risk factors of hypocalcaemia.

  11. miR-33-5p, a novel mechano-sensitive microRNA promotes osteoblast differentiation by targeting Hmga2

    PubMed Central

    Wang, Han; Sun, Zhongyang; Wang, Yixuan; Hu, Zebing; Zhou, Hua; Zhang, Lianchang; Hong, Bo; Zhang, Shu; Cao, Xinsheng

    2016-01-01

    MicroRNAs (miRNAs) interfere with the translation of specific target mRNAs and are thought to thereby regulate many cellular processes. However, the role of miRNAs in osteoblast mechanotransduction remains to be defined. In this study, we investigated the ability of a miRNA to respond to different mechanical environments and regulate mechano-induced osteoblast differentiation. First, we demonstrated that miR-33-5p expressed by osteoblasts is sensitive to multiple mechanical environments, microgravity and fluid shear stress. We then confirmed the ability of miR-33-5p to promote osteoblast differentiation. Microgravity or fluid shear stress influences osteoblast differentiation partially via miR-33-5p. Through bioinformatics analysis and a luciferase assay, we subsequently confirmed that Hmga2 is a target gene of miR-33-5p that negatively regulates osteoblast differentiation. Moreover, miR-33-5p regulates osteoblast differentiation partially via Hmga2. In summary, our findings demonstrate that miR-33-5p is a novel mechano-sensitive miRNA that can promote osteoblast differentiation and participate in the regulation of differentiation induced by changes in the mechanical environment, suggesting this miRNA as a potential target for the treatment of pathological bone loss. PMID:26980276

  12. Identification and proteomic analysis of osteoblast-derived exosomes.

    PubMed

    Ge, Min; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng

    2015-11-01

    Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786 proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases. PMID:26420226

  13. Identification and proteomic analysis of osteoblast-derived exosomes.

    PubMed

    Ge, Min; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng

    2015-11-01

    Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786 proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases.

  14. The LIM protein LIMD1 influences osteoblast differentiation and function

    SciTech Connect

    Luderer, Hilary F.; Bai Shuting; Longmore, Gregory D.

    2008-09-10

    The balance between bone resorption and bone formation involves the coordinated activities of osteoblasts and osteoclasts. Communication between these two cell types is essential for maintenance of normal bone homeostasis; however, the mechanisms regulating this cross talk are not completely understood. Many factors that mediate differentiation and function of both osteoblasts and osteoclasts have been identified. The LIM protein Limd1 has been implicated in the regulation of stress osteoclastogenesis through an interaction with the p62/sequestosome protein. Here we show that Limd1 also influences osteoblast progenitor numbers, differentiation, and function. Limd1{sup -/-} calvarial osteoblasts display increased mineralization and accelerated differentiation. While no significant differences in osteoblast number or function were detected in vivo, bone marrow stromal cells isolated from Limd1{sup -/-} mice contain significantly more osteoblast progenitors compared to wild type controls when cultured ex vivo. Furthermore, we observed a significant increase in nuclear {beta}-catenin staining in differentiating Limd1{sup -/-} calvarial osteoblasts suggesting that Limd1 is a negative regulator of canonical Wnt signaling in osteoblasts. These results demonstrate that Limd1 influences not only stress osteoclastogenesis but also osteoblast function and osteoblast progenitor commitment. Together, these data identify Limd1 as a novel regulator of both bone osetoclast and bone osteoblast development and function.

  15. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation.

    PubMed

    Glass, Donald A; Bialek, Peter; Ahn, Jong Deok; Starbuck, Michael; Patel, Millan S; Clevers, Hans; Taketo, Mark M; Long, Fanxin; McMahon, Andrew P; Lang, Richard A; Karsenty, Gerard

    2005-05-01

    Inactivation of beta-catenin in mesenchymal progenitors prevents osteoblast differentiation; inactivation of Lrp5, a gene encoding a likely Wnt coreceptor, results in low bone mass (osteopenia) by decreasing bone formation. These observations indicate that Wnt signaling controls osteoblast differentiation and suggest that it may regulate bone formation in differentiated osteoblasts. Here, we study later events and find that stabilization of beta-catenin in differentiated osteoblasts results in high bone mass, while its deletion from differentiated osteoblasts leads to osteopenia. Surprisingly, histological analysis showed that these mutations primarily affect bone resorption rather than bone formation. Cellular and molecular studies showed that beta-catenin together with TCF proteins regulates osteoblast expression of Osteoprotegerin, a major inhibitor of osteoclast differentiation. These findings demonstrate that beta-catenin, and presumably Wnt signaling, promote the ability of differentiated osteoblasts to inhibit osteoclast differentiation; thus, they broaden our knowledge of the functions Wnt proteins have at various stages of skeletogenesis. PMID:15866165

  16. Effects of microgravity on osteoblast growth activation.

    PubMed

    Hughes-Fulford, M; Lewis, M L

    1996-04-10

    Space flight is an environmental condition where astronauts can lose up to 19% of weight-bearing bone during long duration missions. We used the MC3T3-E1 osteoblast to investigate bone cell growth in microgravity (10(-6) to 10(-9)g). Osteoblasts were launched on the STS-56 shuttle flight in a quiescent state with 0.5% fetal calf serum (FCS) medium and growth activation was initiated by adding fresh medium with 10% FCS during microgravity exposure. Four days after serum activation, the cells were fixed before return to normal Earth gravity. Ground controls were treated in parallel with the flight samples in identical equipment. On landing, cell number, cell cytoskeleton, glucose utilization, and prostaglandin synthesis in flight (n = 4) and ground controls (n = 4) were examined. The flown osteoblasts grew slowly in microgravity with total cell number significantly reduced (55 +/- 6 vs 141 +/- 8 cells per microscopic field). The cytoskeleton of the flight osteoblasts had a reduced number of stress fibers and a unique abnormal morphology. Nuclei in the ground controls were large and round with punctate Hoechst staining of the DNA nucleosomes. The flight nuclei were 30% smaller than the controls (P < 0.0001) and oblong in shape, with fewer punctate areas. Due to their reduced numbers, the cells activated in microgravity used significantly less glucose than ground controls (80.2 +/- 0.7 vs 50.3 +/- 3.7 mg of glucose/dl remaining in the medium) and had reduced prostaglandin E2 (PGE2) synthesis when compared to controls (57.3 +/- 17 vs 138.3 +/- 41 pmol/ml). Cell viability was normal since, on a per-cell basis, glucose use and prostaglandin synthesis were comparable for flight and ground samples. Taken together, these data suggest that growth activation in microgravity results in reduced growth, causing reduced glucose utilization and reduced prostaglandin synthesis, with significantly altered actin cytoskeleton in osteoblasts.

  17. Effects of microgravity on osteoblast growth activation

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Lewis, M. L.

    1996-01-01

    Space flight is an environmental condition where astronauts can lose up to 19% of weight-bearing bone during long duration missions. We used the MC3T3-E1 osteoblast to investigate bone cell growth in microgravity (10(-6) to 10(-9)g). Osteoblasts were launched on the STS-56 shuttle flight in a quiescent state with 0.5% fetal calf serum (FCS) medium and growth activation was initiated by adding fresh medium with 10% FCS during microgravity exposure. Four days after serum activation, the cells were fixed before return to normal Earth gravity. Ground controls were treated in parallel with the flight samples in identical equipment. On landing, cell number, cell cytoskeleton, glucose utilization, and prostaglandin synthesis in flight (n = 4) and ground controls (n = 4) were examined. The flown osteoblasts grew slowly in microgravity with total cell number significantly reduced (55 +/- 6 vs 141 +/- 8 cells per microscopic field). The cytoskeleton of the flight osteoblasts had a reduced number of stress fibers and a unique abnormal morphology. Nuclei in the ground controls were large and round with punctate Hoechst staining of the DNA nucleosomes. The flight nuclei were 30% smaller than the controls (P < 0.0001) and oblong in shape, with fewer punctate areas. Due to their reduced numbers, the cells activated in microgravity used significantly less glucose than ground controls (80.2 +/- 0.7 vs 50.3 +/- 3.7 mg of glucose/dl remaining in the medium) and had reduced prostaglandin E2 (PGE2) synthesis when compared to controls (57.3 +/- 17 vs 138.3 +/- 41 pmol/ml). Cell viability was normal since, on a per-cell basis, glucose use and prostaglandin synthesis were comparable for flight and ground samples. Taken together, these data suggest that growth activation in microgravity results in reduced growth, causing reduced glucose utilization and reduced prostaglandin synthesis, with significantly altered actin cytoskeleton in osteoblasts.

  18. Bacteria and osteoblast adhesion to chitosan immobilized titanium surface: A race for the surface.

    PubMed

    Foss, Berit L; Ghimire, Niranjan; Tang, Ruogu; Sun, Yuyu; Deng, Ying

    2015-10-01

    In order to evaluate the anti-infective efficacy of the titanium implant materials, two co-culture systems, a low-bacteria/osteoblast (L-B) and a high-bacteria/osteoblast system (H-B), were established. Untreated (UN-Ti), sulfuric acid-treated (SA-Ti), and chitosan immobilized titanium (SA-CS-Ti) materials were developed and evaluated. Bacteria and osteoblast behaviors, including initial attachment (evaluated at 30 mins), adhesion (evaluated at 4 h), and osteoblast spreading on each material surface were evaluated using quantification assays, scanning electron microscopy (SEM), and confocal microscopy. Quantification analysis at 30 mins showed significantly higher number of osteoblast present on SA-CS-Ti in both L-B (10,083 ± 2626) and H-B (23,592 ± 2233) than those on the UN-Ti (p<0.05). SEM observation and confocal microscopy results showed more surface area was occupied by adhered osteoblasts on SA-CS-Ti than UN-Ti and SA-Ti in both co-culture systems at 30 mins. At all time points, SA-CS-Ti had the lowest level of bacterial adhesion compared to UN-Ti and SA-Ti in both co-culture systems. A significantly (p<0.05) lower number of bacteria were recovered from SA-CS-Ti (2233 ± 681) in the H-B system compared to UN-Ti (5367 ± 1662) and SA-Ti (4533 ± 680) at 4h. Quantitative and qualitative co-culture results show the great potential of chitosan immobilization onto implant materials to prevent implant-associated infections. PMID:26222405

  19. Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism

    PubMed Central

    Allen, Kahtonna C.; Sanchez, Carlos J.; Niece, Krista L.; Wenke, Joseph C.

    2015-01-01

    Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity. PMID:26324277

  20. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

    PubMed Central

    Pellegrini, Gretel G.; Morales, Cynthya C.; Wallace, Taylor C.; Plotkin, Lilian I.; Bellido, Teresita

    2016-01-01

    Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further

  1. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner.

    PubMed

    Pellegrini, Gretel G; Morales, Cynthya C; Wallace, Taylor C; Plotkin, Lilian I; Bellido, Teresita

    2016-01-01

    Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further

  2. Effects of Polypropylene Carbonate/Poly(D,L-lactic) Acid/Tricalcium Phosphate Elastic Composites on Improving Osteoblast Maturation.

    PubMed

    Fang, Hsu-Wei; Kao, Wei-Yu; Lin, Pei-I; Chang, Guang-Wei; Hung, Ya-Jung; Chen, Ruei-Ming

    2015-08-01

    Bone tissue engineering utilizing biomaterials to improve osteoblast growth has provided de novo consideration for therapy of bone diseases. Polypropylene carbonate (PPC) is a polymer with a low glass transition temperature but high elasticity. In this study, we developed a new PPC-derived composite by mixing poly-lactic acid (PLA) and tricalcium phosphate (TCP), called PPC/PLA/TCP elastic (PPTE) scaffolds. We also evaluated the beneficial effects of PPTE composites on osteoblast growth and maturation and the possible mechanisms. Compared to PPC polymers, PPTE composites had similar pore sizes and porosities but possessed better hydrophilic surface structures. Biological evaluations further revealed that PPTE composites attracted adhesion of mouse osteoblasts, and these bone cells extended along the porous scaffolds to produce accurate fibroblast-like morphologies. In parallel, seeding mouse osteoblasts onto PPTE composites time-dependently increased cell growth. Sequentially, PPTE composites augmented DNA replication and cell proliferation. Consequently, PPTE composites significantly improved osteoblast mineralization. As to the mechanism, treatment with PPTE composites induced osteopontin (OPN) mRNA and protein expression and alkaline phosphatase activity. Taken together, this study showed that PPTE composites with porous and hydrophilic surfaces can stimulate osteoblast adhesion, proliferation, and maturation through an OPN-dependent mechanism. Therefore, the de novo PPTE scaffolds may have biomaterial potential for bone regeneration. PMID:25549776

  3. Autophagy mediated CoCrMo particle-induced peri-implant osteolysis by promoting osteoblast apoptosis.

    PubMed

    Wang, Zhenheng; Liu, Naicheng; Liu, Kang; Zhou, Gang; Gan, Jingjing; Wang, Zhenzhen; Shi, Tongguo; He, Wei; Wang, Lintao; Guo, Ting; Bao, Nirong; Wang, Rui; Huang, Zhen; Chen, Jiangning; Dong, Lei; Zhao, Jianning; Zhang, Junfeng

    2015-01-01

    Wear particle-induced osteolysis is the leading cause of aseptic loosening, which is the most common reason for THA (total hip arthroplasty) failure and revision surgery. Although existing studies suggest that osteoblast apoptosis induced by wear debris is involved in aseptic loosening, the underlying mechanism linking wear particles to osteoblast apoptosis remains almost totally unknown. In the present study, we investigated the effect of autophagy on osteoblast apoptosis induced by CoCrMo metal particles (CoPs) in vitro and in a calvarial resorption animal model. Our study demonstrated that CoPs stimulated autophagy in osteoblasts and PIO (particle-induced osteolysis) animal models. Both autophagy inhibitor 3-MA (3-methyladenine) and siRNA of Atg5 could dramatically reduce CoPs-induced apoptosis in osteoblasts. Further, inhibition of autophagy with 3-MA ameliorated the severity of osteolysis in PIO animal models. Moreover, 3-MA also prevented osteoblast apoptosis in an antiautophagic way when tested in PIO model. Collectively, these results suggest that autophagy plays a key role in CoPs-induced osteolysis and that targeting autophagy-related pathways may represent a potential therapeutic approach for treating particle-induced peri-implant osteolysis.

  4. Effects of Polypropylene Carbonate/Poly(D,L-lactic) Acid/Tricalcium Phosphate Elastic Composites on Improving Osteoblast Maturation.

    PubMed

    Fang, Hsu-Wei; Kao, Wei-Yu; Lin, Pei-I; Chang, Guang-Wei; Hung, Ya-Jung; Chen, Ruei-Ming

    2015-08-01

    Bone tissue engineering utilizing biomaterials to improve osteoblast growth has provided de novo consideration for therapy of bone diseases. Polypropylene carbonate (PPC) is a polymer with a low glass transition temperature but high elasticity. In this study, we developed a new PPC-derived composite by mixing poly-lactic acid (PLA) and tricalcium phosphate (TCP), called PPC/PLA/TCP elastic (PPTE) scaffolds. We also evaluated the beneficial effects of PPTE composites on osteoblast growth and maturation and the possible mechanisms. Compared to PPC polymers, PPTE composites had similar pore sizes and porosities but possessed better hydrophilic surface structures. Biological evaluations further revealed that PPTE composites attracted adhesion of mouse osteoblasts, and these bone cells extended along the porous scaffolds to produce accurate fibroblast-like morphologies. In parallel, seeding mouse osteoblasts onto PPTE composites time-dependently increased cell growth. Sequentially, PPTE composites augmented DNA replication and cell proliferation. Consequently, PPTE composites significantly improved osteoblast mineralization. As to the mechanism, treatment with PPTE composites induced osteopontin (OPN) mRNA and protein expression and alkaline phosphatase activity. Taken together, this study showed that PPTE composites with porous and hydrophilic surfaces can stimulate osteoblast adhesion, proliferation, and maturation through an OPN-dependent mechanism. Therefore, the de novo PPTE scaffolds may have biomaterial potential for bone regeneration.

  5. Autophagy mediated CoCrMo particle-induced peri-implant osteolysis by promoting osteoblast apoptosis

    PubMed Central

    Wang, Zhenheng; Liu, Naicheng; Liu, Kang; Zhou, Gang; Gan, Jingjing; Wang, Zhenzhen; Shi, Tongguo; He, Wei; Wang, Lintao; Guo, Ting; Bao, Nirong; Wang, Rui; Huang, Zhen; Chen, Jiangning; Dong, Lei; Zhao, Jianning; Zhang, Junfeng

    2015-01-01

    Wear particle-induced osteolysis is the leading cause of aseptic loosening, which is the most common reason for THA (total hip arthroplasty) failure and revision surgery. Although existing studies suggest that osteoblast apoptosis induced by wear debris is involved in aseptic loosening, the underlying mechanism linking wear particles to osteoblast apoptosis remains almost totally unknown. In the present study, we investigated the effect of autophagy on osteoblast apoptosis induced by CoCrMo metal particles (CoPs) in vitro and in a calvarial resorption animal model. Our study demonstrated that CoPs stimulated autophagy in osteoblasts and PIO (particle-induced osteolysis) animal models. Both autophagy inhibitor 3-MA (3-methyladenine) and siRNA of Atg5 could dramatically reduce CoPs-induced apoptosis in osteoblasts. Further, inhibition of autophagy with 3-MA ameliorated the severity of osteolysis in PIO animal models. Moreover, 3-MA also prevented osteoblast apoptosis in an antiautophagic way when tested in PIO model. Collectively, these results suggest that autophagy plays a key role in CoPs-induced osteolysis and that targeting autophagy-related pathways may represent a potential therapeutic approach for treating particle-induced peri-implant osteolysis. PMID:26566231

  6. Effects of Porphyromonas gingivalis lipopolysaccharide on osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling

    PubMed Central

    ZHANG, YI; WANG, XI-CHAO; BAO, XING-FU; HU, MIN; YU, WEI-XIAN

    2014-01-01

    In bone remodeling, the Eph family is involved in regulating the process of osteoclast and osteoblast coordination in order to maintain bone homeostasis. In this study, the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling were investigated. An osteoblast-osteoclast co-culture system was achieved successfully. Hence, direct contact and communication between osteoblasts and osteoclasts was permitted. Regarding the protein expression and gene expression of EphB4 and EphrinB2, it was shown that Pg-LPS increased the expression of EphB4 while inhibiting the expression of EphrinB2. Therefore, the results indicate that, when treated with Pg-LPS, the EphB4 receptor on osteoblasts and the EphrinB2 ligand on osteoclasts may generate bidirectional anti-osteoclastogenic and pro-osteoblastogenic signaling into respective cells and potentially facilitate the transition from bone resorption to bone formation. This study may contribute to the control of osteoblast differentiation and bone formation at remodeling, and possibly also modeling, sites. PMID:24348768

  7. Effects of Porphyromonas gingivalis lipopolysaccharide on osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling.

    PubMed

    Zhang, Yi; Wang, Xi-Chao; Bao, Xing-Fu; Hu, Min; Yu, Wei-Xian

    2014-01-01

    In bone remodeling, the Eph family is involved in regulating the process of osteoclast and osteoblast coordination in order to maintain bone homeostasis. In this study, the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling were investigated. An osteoblast-osteoclast co-culture system was achieved successfully. Hence, direct contact and communication between osteoblasts and osteoclasts was permitted. Regarding the protein expression and gene expression of EphB4 and EphrinB2, it was shown that Pg-LPS increased the expression of EphB4 while inhibiting the expression of EphrinB2. Therefore, the results indicate that, when treated with Pg-LPS, the EphB4 receptor on osteoblasts and the EphrinB2 ligand on osteoclasts may generate bidirectional anti-osteoclastogenic and pro-osteoblastogenic signaling into respective cells and potentially facilitate the transition from bone resorption to bone formation. This study may contribute to the control of osteoblast differentiation and bone formation at remodeling, and possibly also modeling, sites.

  8. Reactive polyurethane carbon nanotube foams and their interactions with osteoblasts.

    PubMed

    Verdejo, Raquel; Jell, Gavin; Safinia, Laleh; Bismarck, Alexander; Stevens, Molly M; Shaffer, Milo S P

    2009-01-01

    The remarkable intrinsic properties of carbon nanotubes, including their high mechanical strength, electrical conductivity, and nanoscale 3D architecture, create promising opportunities for the use of nanotube composites in a number of fields, particularly for composites in which conventional fillers cannot be accommodated. In the current study, 3D polyurethane (PU) nanocomposite foams were developed, and their potential biomedical applications were investigated. Multiwalled carbon nanotubes (CNTs) were synthesized by chemical vapor deposition and, following suitable chemical modification, uniformly distributed within the walls of PU foams produced by direct reaction. Although the loading fraction was too low to observe significant mechanical effects, CNT incorporation improved the wettability of the nanocomposite surfaces in a concentration-dependent manner, supporting the claim that the nanotubes are active at the pore surface. Studies of bone cell interactions with the nanocomposite foams revealed that increasing CNT loading fraction did not cause osteoblast cytotoxicity nor have any detrimental effects on osteoblast differentiation or mineralization. The application of "fixed" or embedded CNTs in nondegradable scaffolds is likely advantageous over "loose" or unattached CNTs from a toxicological point of view.

  9. Optical diagnostics of osteoblast cells and osteogenic drug screening

    NASA Astrophysics Data System (ADS)

    Kolanti, Elayaraja; Veerla, Sarath C.; Khajuria, Deepak K.; Roy Mahapatra, D.

    2016-02-01

    Microfluidic device based diagnostics involving optical fibre path, in situ imaging and spectroscopy are gaining importance due to recent advances in diagnostics instrumentation and methods, besides other factors such as low amount of reagent required for analysis, short investigation times, and potential possibilities to replace animal model based study in near future. It is possible to grow and monitor tissues in vitro in microfluidic lab-on-chip. It may become a transformative way of studying how cells interact with drugs, pathogens and biomaterials in physiologically relevant microenvironments. To a large extent, progress in developing clinically viable solutions has been constrained because of (i) contradiction between in vitro and in vivo results and (ii) animal model based and clinical studies which is very expensive. Our study here aims to evaluate the usefulness of microfluidic device based 3D tissue growth and monitoring approach to better emulate physiologically and clinically relevant microenvironments in comparison to conventional in vitro 2D culture. Moreover, the microfluidic methodology permits precise high-throughput investigations through real-time imaging while using very small amounts of reagents and cells. In the present study, we report on the details of an osteoblast cell based 3D microfluidic platform which we employ for osteogenic drug screening. The drug formulation is functionalized with fluorescence and other biomarkers for imaging and spectroscopy, respectively. Optical fibre coupled paths are used to obtain insight regarding the role of stress/flow pressure fluctuation and nanoparticle-drug concentration on the osteoblast growth and osteogenic properties of bone.

  10. Role of integrin α2 β1 in mediating osteoblastic differentiation on three-dimensional titanium scaffolds with submicron-scale texture.

    PubMed

    Wang, Xiaokun; Schwartz, Zvi; Gittens, Rolando A; Cheng, Alice; Olivares-Navarrete, Rene; Chen, Haifeng; Boyan, Barbara D

    2015-06-01

    Hierarchical surface roughness of titanium and titanium alloy implants plays an important role in osseointegration. In vitro and in vivo studies show greater osteoblast differentiation and bone formation when implants have submicron-scale textured surfaces. In this study, we tested the potential benefit of combining a submicron-scale textured surface with three-dimensional (3D) structure on osteoblast differentiation and the involvement of an integrin-driven mechanism. 3D titanium scaffolds were made using orderly oriented titanium meshes and microroughness was added to the wire surface by acid-etching. MG63 and human osteoblasts were seeded on 3D scaffolds and 2D surfaces with or without acid etching. At confluence, increased osteocalcin, vascular endothelial growth factor, osteoprotegerin (OPG), and alkaline phosphatase (ALP) activity were observed in MG63 and human osteoblasts on 3D scaffolds in comparison to 2D surfaces at the protein level, indicating enhanced osteoblast differentiation. To further investigate the mechanism of osteoblast-3D scaffold interaction, the role of integrin α2β1 was examined. The results showed β1 and α2β1 integrin silencing abolished the increase in osteoblastic differentiation markers on 3D scaffolds. Time course studies showed osteoblasts matured faster in the 3D environment in the early stage of culture, while as cells proliferated, the maturation slowed down to a comparative level as 2D surfaces. After 12 days of postconfluent culture, osteoblasts on 3D scaffolds showed a second-phase increase in ALP activity. This study shows that osteoblastic differentiation is improved on 3D scaffolds with submicron-scale texture and is mediated by integrin α2β1.

  11. Role of integrin α2β1 in mediating osteoblastic differentiation on three-dimensional titanium scaffolds with submicron-scale texture

    PubMed Central

    Wang, Xiaokun; Schwartz, Zvi; Gittens, Rolando A.; Cheng, Alice; Olivares-Navarrete, Rene; Chen, Haifeng; Boyan, Barbara D.

    2014-01-01

    Hierarchical surface roughness of titanium and titanium alloy implants plays an important role in osseointegration. In vitro and in vivo studies show greater osteoblast differentiation and bone formation when implants have submicron-scale textured surfaces. In this study, we tested the potential benefit of combining a submicron-scale textured surface with three-dimensional (3D) structure on osteoblast differentiation and the involvement of an integrin-driven mechanism. 3D titanium scaffolds were made using orderly oriented titanium meshes and microroughness was added to the wire surface by acid-etching. MG63 and human osteoblasts were seeded on 3D scaffolds and 2D surfaces with or without acid etching. At confluence, increased osteocalcin, vascular endothelial growth factor, osteoprotegerin (OPG), and alkaline phosphatase (ALP) activity were observed in MG63 and human osteoblasts on 3D scaffolds in comparison to 2D surfaces at the protein level, indicating enhanced osteoblast differentiation. To further investigate the mechanism of osteoblast-3D scaffold interaction, the role of integrin α2β1 was examined. The results showed β1 and α2β1 integrin silencing abolished the increase in osteoblastic differentiation markers on 3D scaffolds. Time course studies showed osteoblasts matured faster in the 3D environment in the early stage of culture, while as cells proliferated, the maturation slowed down to a comparative level as 2D surfaces. After 12 days of postconfluent culture, osteoblasts on 3D scaffolds showed a second-phase increase in ALP activity. This study shows that osteoblastic differentiation is improved on 3D scaffolds with submicron-scale texture and is mediated by integrin α2β1. PMID:25203434

  12. Loss of Osteoblast Runx3 Produces Severe Congenital Osteopenia

    PubMed Central

    Bauer, Omri; Sharir, Amnon; Kimura, Ayako; Hantisteanu, Shay; Takeda, Shu

    2015-01-01

    Congenital osteopenia is a bone demineralization condition that is associated with elevated fracture risk in human infants. Here we show that Runx3, like Runx2, is expressed in precommitted embryonic osteoblasts and that Runx3-deficient mice develop severe congenital osteopenia. Runx3-deficient osteoblast-specific (Runx3fl/fl/Col1α1-cre), but not chondrocyte-specific (Runx3fl/fl/Col1α2-cre), mice are osteopenic. This demonstrates that an osteoblastic cell-autonomous function of Runx3 is required for proper osteogenesis. Bone histomorphometry revealed that decreased osteoblast numbers and reduced mineral deposition capacity in Runx3-deficient mice cause this bone formation deficiency. Neonatal bone and cultured primary osteoblast analyses revealed a Runx3-deficiency-associated decrease in the number of active osteoblasts resulting from diminished proliferation and not from enhanced osteoblast apoptosis. These findings are supported by Runx3-null culture transcriptome analyses showing significant decreases in the levels of osteoblastic markers and increases in the levels of Notch signaling components. Thus, while Runx2 is mandatory for the osteoblastic lineage commitment, Runx3 is nonredundantly required for the proliferation of these precommitted cells, to generate adequate numbers of active osteoblasts. Human RUNX3 resides on chromosome 1p36, a region that is associated with osteoporosis. Therefore, RUNX3 might also be involved in human bone mineralization. PMID:25605327

  13. Effects of microgravity on osteoblast growth.

    PubMed

    Hughes-Fulford, M; Tjandrawinata, R; Fitzgerald, J; Gasuad, K; Gilbertson, V

    1998-05-01

    Studies from space flights over the past two decades have demonstrated that basic physiological changes occur in humans during space flight. These changes include cephalic fluid shifts, loss of fluid and electrolytes, loss of muscle mass, space motion sickness, anemia, reduced immune response, and loss of calcium and mineralized bone. The cause of most of these manifestations is not known and until recently, the general approach was to investigate general systemic changes, not basic cellular responses to microgravity. Recently analyzed data from the 1973-1974 Skylabs disclose that there is a rise in the systemic hormone, cortisol, which may play a role in bone loss in flight. In two flights where bone growth was measured (Skylabs 3 and 4), the crew members had a significant loss of calcium accompanied by a rise in 24 hour urinary cortisol during the entire flight period. In ground-based work on osteoblasts, we have demonstrated that equivalent amounts of glucocorticoids can inhibit osteoblast cell growth. In addition, this laboratory has recently studied gene growth and activation of mouse osteoblasts (MC3T3-E1) during spaceflight. Osteoblast cells were grown on glass coverslips, loaded in the Biorack plunger boxes 18 hours before launch and activated 19 hours after launch in the Biorack incubator under microgravity conditions. The osteoblasts were launched in a serum deprived state, activated and collected in microgravity. Samples were collected at 29 hours after sera activation (0-g, n=4; 1-g, n=4). The osteoblasts were examined for changes in gene expression and cell morphology. Approximately one day after growth activation, remarkable differences were observed in gene expression in 0-g and 1-g flight samples. The 0-g activated cells had increased c-fos mRNA when compared to flight 1-g controls. The message of immediate early growth gene, cox-2 was decreased in the microgravity activated cells when compared to ground or 1-g flight controls. Cox-1 was not

  14. Effects of microgravity on osteoblast growth

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Tjandrawinata, R.; Fitzgerald, J.; Gasuad, K.; Gilbertson, V.

    1998-01-01

    Studies from space flights over the past two decades have demonstrated that basic physiological changes occur in humans during space flight. These changes include cephalic fluid shifts, loss of fluid and electrolytes, loss of muscle mass, space motion sickness, anemia, reduced immune response, and loss of calcium and mineralized bone. The cause of most of these manifestations is not known and until recently, the general approach was to investigate general systemic changes, not basic cellular responses to microgravity. Recently analyzed data from the 1973-1974 Skylabs disclose that there is a rise in the systemic hormone, cortisol, which may play a role in bone loss in flight. In two flights where bone growth was measured (Skylabs 3 and 4), the crew members had a significant loss of calcium accompanied by a rise in 24 hour urinary cortisol during the entire flight period. In ground-based work on osteoblasts, we have demonstrated that equivalent amounts of glucocorticoids can inhibit osteoblast cell growth. In addition, this laboratory has recently studied gene growth and activation of mouse osteoblasts (MC3T3-E1) during spaceflight. Osteoblast cells were grown on glass coverslips, loaded in the Biorack plunger boxes 18 hours before launch and activated 19 hours after launch in the Biorack incubator under microgravity conditions. The osteoblasts were launched in a serum deprived state, activated and collected in microgravity. Samples were collected at 29 hours after sera activation (0-g, n=4; 1-g, n=4). The osteoblasts were examined for changes in gene expression and cell morphology. Approximately one day after growth activation, remarkable differences were observed in gene expression in 0-g and 1-g flight samples. The 0-g activated cells had increased c-fos mRNA when compared to flight 1-g controls. The message of immediate early growth gene, cox-2 was decreased in the microgravity activated cells when compared to ground or 1-g flight controls. Cox-1 was not

  15. BK Knockout by TALEN-Mediated Gene Targeting in Osteoblasts: KCNMA1 Determines the Proliferation and Differentiation of Osteoblasts

    PubMed Central

    Hei, Hongya; Gao, Jianjun; Dong, Jibin; Tao, Jie; Tian, Lulu; Pan, Wanma; Wang, Hongyu; Zhang, Xuemei

    2016-01-01

    Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BK-knockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases. PMID:27329042

  16. BK Knockout by TALEN-Mediated Gene Targeting in Osteoblasts: KCNMA1 Determines the Proliferation and Differentiation of Osteoblasts.

    PubMed

    Hei, Hongya; Gao, Jianjun; Dong, Jibin; Tao, Jie; Tian, Lulu; Pan, Wanma; Wang, Hongyu; Zhang, Xuemei

    2016-07-01

    Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BK-knockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases.

  17. Estrogen Regulation of Apoptosis in Osteoblasts

    PubMed Central

    Bradford, Peter G; Gerace, Ken V; Roland, Renée L; Chrzan, Brian G

    2010-01-01

    Dysregulated apoptosis is a critical failure associated with prominent degenerative diseases including osteoporosis. In bone, estrogen deficiency has been associated with accelerated osteoblast apoptosis and susceptibility to osteoporotic fractures. Hormone therapy continues to be an effective option for preventing osteoporosis and bone fractures. Induction of apoptosis in G-292 human osteoblastic cells by exposure to etoposide or the inflammatory cytokine TNFα promoted acute caspase-3/7 activity and this increased activity was inhibited by pretreatment with estradiol. Etoposide also increased the expression of a battery of apoptosis-promoting genes and this expression was also inhibited by estradiol. Among the apoptotic genes whose expression was inhibited by estradiol was ITPR1, which encodes the type 1 InsP3R. InsP3Rs are intracellular calcium channels and key proapoptotic mediators. Estradiol via estrogen receptor β1 suppresses ITPR1 gene transcription in G-292 cells. These analyses suggest that an underlying basis of the beneficial activity of estrogens in combating osteoporosis may involve the prevention of apoptosis in osteoblasts and that a key event in this process is the repression of apoptotic gene expression and inhibition of caspase-3/7. PMID:19426747

  18. p38 MAPK Signaling in Osteoblast Differentiation

    PubMed Central

    Rodríguez-Carballo, Eddie; Gámez, Beatriz; Ventura, Francesc

    2016-01-01

    The skeleton is a highly dynamic tissue whose structure relies on the balance between bone deposition and resorption. This equilibrium, which depends on osteoblast and osteoclast functions, is controlled by multiple factors that can be modulated post-translationally. Some of the modulators are Mitogen-activated kinases (MAPKs), whose role has been studied in vivo and in vitro. p38-MAPK modifies the transactivation ability of some key transcription factors in chondrocytes, osteoblasts and osteoclasts, which affects their differentiation and function. Several commercially available inhibitors have helped to determine p38 action on these processes. Although it is frequently mentioned in the literature, this chemical approach is not always as accurate as it should be. Conditional knockouts are a useful genetic tool that could unravel the role of p38 in shaping the skeleton. In this review, we will summarize the state of the art on p38 activity during osteoblast differentiation and function, and emphasize the triggers of this MAPK. PMID:27200351

  19. Staphylococcus aureus vs. Osteoblast: Relationship and Consequences in Osteomyelitis

    PubMed Central

    Josse, Jérôme; Velard, Frédéric; Gangloff, Sophie C.

    2015-01-01

    Bone cells, namely osteoblasts and osteoclasts work in concert and are responsible for bone extracellular matrix formation and resorption. This homeostasis is, in part, altered during infections by Staphylococcus aureus through the induction of various responses from the osteoblasts. This includes the over-production of chemokines, cytokines and growth factors, thus suggesting a role for these cells in both innate and adaptive immunity. S. aureus decreases the activity and viability of osteoblasts, by induction of apoptosis-dependent and independent mechanisms. The tight relationship between osteoclasts and osteoblasts is also modulated by S. aureus infection. The present review provides a survey of the relevant literature discussing the important aspects of S. aureus and osteoblast interaction as well as the ability for antimicrobial peptides to kill intra-osteoblastic S. aureus, hence emphasizing the necessity for new anti-infectious therapeutics. PMID:26636047

  20. Lysyl Oxidase (Lox) Gene Deficiency Affects Osteoblastic Phenotype

    PubMed Central

    Pischon, N.; Mäki, J. M.; Weisshaupt, P.; Heng, N.; Palamakumbura, A. H.; N'Guessan, P.; Ding, A.; Radlanski, R.; Renz, H.; Bronckers, T. A. L. J. J.; Myllyharju, J.; Kielbassa, A.; Kleber, B. M.; Bernimoulin, J.-P.; Trackman, P.C.

    2010-01-01

    Lysyl oxidase (LOX) catalyzes cross-linking of elastin and collagen, which is essential for structural integrity and function of bone tissue. The present study examined the role of Lox gene deficiency for the osteoblast phenotype in primary calvarial osteoblasts from E18.5 Lox knockout (Lox-/-) and wild type (wt) (C57 BL/6) mice. Next to Lox gene depletion, mRNA expression of Lox isoforms, LOXL1-4, was significantly down-regulated in Lox-/- bone tissue. A significant decrease of DNA synthesis of Lox-/- osteoblasts compared to wt was found. Early stages of osteoblastic apoptosis studied by Annexin-V binding as well as later stages of DNA fragmentation were not affected. However, mineral nodule formation and osteoblastic differentiation were markedly decreased, as revealed by significant down-regulation of osteoblastic markers, type I collagen, BSP and Runx2/Cbfa1. PMID:19458888

  1. Insulin signaling in osteoblasts integrates bone remodeling and energy metabolism

    PubMed Central

    Ferron, Mathieu; Wei, Jianwen; Yoshizawa, Tatsuya; Fattore, Andrea Del; DePinho, Ronald A.; Teti, Anna; Ducy, Patricia; Karsenty, Gerard

    2010-01-01

    The broad expression of the insulin receptor suggests that the spectrum of insulin function has not been fully described. A cell type expressing this receptor is the osteoblast, a bone-specific cell favoring glucose metabolism through a hormone, osteocalcin, that becomes active once uncarboxylated. We show here that insulin signaling in osteoblasts is necessary for whole-body glucose homeostasis because it increases osteocalcin activity. To achieve this function insulin signaling in osteoblasts takes advantage of the regulation of osteoclastic bone resorption exerted by osteoblasts. Indeed, since bone resorption occurs at a pH acid enough to decarboxylate proteins, osteoclasts determine the carboxylation status and function of osteocalcin. Accordingly, increasing or decreasing insulin signaling in osteoblasts promotes or hampers glucose metabolism in a bone resorption-dependent manner in mice and humans. Hence, in a feed-forward loop, insulin signals in osteoblasts to activate a hormone, osteocalcin, that promotes glucose metabolism. PMID:20655470

  2. Parathyroid hormone modulates the response of osteoblast-like cells to mechanical stimulation

    NASA Technical Reports Server (NTRS)

    Ryder, K. D.; Duncan, R. L.

    2000-01-01

    Mechanical loading stimulates many responses in bone and osteoblasts associated with osteogenesis. Since loading and parathyroid hormone (PTH) activate similar signaling pathways in osteoblasts, we postulate that PTH can potentiate the effects of mechanical stimulation. Using an in vitro four-point bending device, we found that expression of COX-2, the inducible isoform of cyclooxygenase, was dependent on fluid forces generated across the culture plate, but not physiologic levels of strain in MC3T3-E1 osteoblast-like cells. Addition of 50 nM PTH during loading increased COX-2 expression at both subthreshold and threshold levels of fluid forces compared with either stimuli alone. We also demonstrated that application of fluid shear to MC3T3-E1 cells induced a rapid increase in [Ca(2+)](i). Although PTH did not significantly change [Ca(2+)](i) levels, flow and PTH did produce a significantly greater [Ca(2+)](i) response and increased the number of responding cells than is found in fluid shear alone. The [Ca(2+)](i) response to these stimuli was significantly decreased when the mechanosensitive channel inhibitor, gadolinium, was present. These studies indicate that PTH increases the cellular responses of osteoblasts to mechanical loading. Furthermore, this response may be mediated by alterations in [Ca(2+)](i) by modulating the mechanosensitive channel.

  3. Titanium With Nanotopography Induces Osteoblast Differentiation by Regulating Endogenous Bone Morphogenetic Protein Expression and Signaling Pathway.

    PubMed

    M S Castro-Raucci, Larissa; S Francischini, Marcelo; N Teixeira, Lucas; P Ferraz, Emanuela; B Lopes, Helena; T de Oliveira, Paulo; Hassan, Mohammad Q; Losa, Adalberto L; Beloti, Marcio M

    2016-07-01

    We aimed at evaluating the effect of titanium (Ti) with nanotopography (Nano) on the endogenous expression of BMP-2 and BMP-4 and the relevance of this process to the nanotopography-induced osteoblast differentiation. MC3T3-E1 cells were grown on Nano and machined (Machined) Ti surfaces and the endogenous BMP-2/4 expression and the effect of BMP receptor BMPR1A silencing in both osteoblast differentiation and expression of genes related to TGF-β/BMP signaling were evaluated. Nano supported higher BMP-2 gene and protein expression and upregulated the osteoblast differentiation compared with Machined Ti surface. The BMPR1A silencing inhibited the osteogenic potential induced by Nano Ti surface as indicated by reduced alkaline phosphatase (ALP), osteocalcin and RUNX2 gene expression, RUNX2 protein expression and ALP activity. In addition, the expression of genes related to TGF-β/BMP signaling was deeply affected by BMPR1A-silenced cells grown on Nano Ti surface. In conclusion, we have demonstrated for the first time that nanotopography induces osteoblast differentiation, at least in part, by upregulating the endogenous production of BMP-2 and modulating BMP signaling pathway. J. Cell. Biochem. 117: 1718-1726, 2016. © 2015 Wiley Periodicals, Inc. PMID:26681207

  4. Effects of microgravity on osteoclast bone resorption and osteoblast cytoskeletal organization and adhesion.

    PubMed

    Nabavi, Noushin; Khandani, Arian; Camirand, Anne; Harrison, Rene E

    2011-11-01

    Exposure to microgravity has been associated with several physiological changes in astronauts, including an osteoporosis-like loss in bone mass. Despite many in vivo and in vitro studies in both microgravity and simulated microgravity conditions, the mechanism for bone loss is still not clear. The lack of weight-bearing forces makes microgravity an ideal physical stimulus to assess bone cell responses. In this work, we conduct a unique investigation of the effects of microgravity on bone-producing osteoblasts and, in parallel, on bone-resorbing osteoclasts. An increase in total number of discrete resorption pits is observed in osteoclasts that experienced microgravity versus ground controls. We further show that osteoblasts exposed to 5 days of microgravity have shorter and wavier microtubules (MTs), smaller and fewer focal adhesions, and thinner cortical actin and stress fibers. Space-flown osteoblasts present extended cell shapes as well as significantly more disrupted and often fragmented or condensed nuclei. The absence of gravitational forces therefore causes both an increase in bone resorption by osteoclasts, and a decrease in osteoblast cellular integrity. The observed effects on both major bone cell types likely accelerate bone loss in microgravity environments, and additionally offer a potential explanation to the development of disuse osteoporosis on Earth. PMID:21839189

  5. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro

    PubMed Central

    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-01-01

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  6. Effects of microgravity on osteoclast bone resorption and osteoblast cytoskeletal organization and adhesion.

    PubMed

    Nabavi, Noushin; Khandani, Arian; Camirand, Anne; Harrison, Rene E

    2011-11-01

    Exposure to microgravity has been associated with several physiological changes in astronauts, including an osteoporosis-like loss in bone mass. Despite many in vivo and in vitro studies in both microgravity and simulated microgravity conditions, the mechanism for bone loss is still not clear. The lack of weight-bearing forces makes microgravity an ideal physical stimulus to assess bone cell responses. In this work, we conduct a unique investigation of the effects of microgravity on bone-producing osteoblasts and, in parallel, on bone-resorbing osteoclasts. An increase in total number of discrete resorption pits is observed in osteoclasts that experienced microgravity versus ground controls. We further show that osteoblasts exposed to 5 days of microgravity have shorter and wavier microtubules (MTs), smaller and fewer focal adhesions, and thinner cortical actin and stress fibers. Space-flown osteoblasts present extended cell shapes as well as significantly more disrupted and often fragmented or condensed nuclei. The absence of gravitational forces therefore causes both an increase in bone resorption by osteoclasts, and a decrease in osteoblast cellular integrity. The observed effects on both major bone cell types likely accelerate bone loss in microgravity environments, and additionally offer a potential explanation to the development of disuse osteoporosis on Earth.

  7. Adipose-derived mesenchymal stromal (stem) cells differentiate to osteoblast and chondroblast lineages upon incubation with conditioned media from dental pulp stem cell-derived osteoblasts and auricle cartilage chondrocytes.

    PubMed

    Carbone, A; Valente, M; Annacontini, L; Castellani, S; Di Gioia, S; Parisi, D; Rucci, M; Belgiovine, G; Colombo, C; Di Benedetto, A; Mori, G; Lo Muzio, L; Maiorella, A; Portincasa, A; Conese, M

    2016-01-01

    The potential of adipose-derived mesenchymal stromal (stem) cells (ADSCs) to differentiate into either osteoblasts or chondrocytes is controversial. In this study we investigated the multicapacity potential of ADSCs to differentiate towards adipocyte, osteoblast, and chondrocyte lineages when cells are seeded onto plastic in comparison with incubation with conditioned media (CM) obtained from differentiated cell types.ADSCs, obtained from liposuctions, were characterized for mesenchymal and hematopoietic markers by cytofluorimetry. Their differentiation capacity towards adipocytes, osteoblasts, and chondrocytes was investigated by histochemistry methods (Oil-Red-O staining, Safranin O and Alizarin Red staining, respectively). Dental pulp stem cells (DPSCs) and dedifferentiated auricle derived-chondrocytes were differentiated towards osteoblastic and chondrocytic lineages respectively, and the CM obtained from these cultures was used to induce differentiation of ADSCs. ADSCs were positive for mesenchymal markers (CD29, CD105, CD73, CD44), but not for hematopoietic lineage markers (CD14, CD34, CD45) and this behavior was conserved from the isolation up to the fifth passage. While ADSCs were readily differentiated in adipocytes, they were not towards chondrocytes and osteoblastic lineages, a behavior different from that of bone marrow-derived MSCs that differentiated into the three lineages at two weeks post-induction. Only ADSCs treated with CM from cultured chondrocytes and DPSCs, produced glycosaminoglycans and mineralized matrix. These results indicate that ADSCs need growth/morphogenic factor supplementation from the tissue environment to be appropriately differentiated to mesodermic lineages. PMID:27049081

  8. Analysis of human alveolar osteoblast behavior on a nano-hydroxyapatite substrate: an in vitro study

    PubMed Central

    2014-01-01

    Background Nano-hydroxyapatite (nHA) is a potential ideal biomaterial for bone regeneration. However, studies have yet to characterize the behavior of human osteoblasts derived from alveolar bone on nHA. Thus, the aim of the present study was to evaluate the influence of nHA on the adhesion, proliferation and differentiation of these alveolar bone-derived cells. Methods Primary human alveolar osteoblasts were collected from the alveolar ridge of a male periodontal patient during osseous resective surgery and grown on culture plates coated with either polylysine or polylysine with nano-hydroxyapatite (POL/nHA) composite. The cells were grown and observed for 14 days, and then assessed for potential modifications to osteoblasts homeostasis as evaluated by quantitative reverse transcriptase-polymerase chain reaction (real time RT-PCR), scanning electron microscopy and atomic force microscopy. Results Real time PCR revealed a significant increase in the expression of the selected markers of osteoblast differentiation (bone morphogenetic protein (BMP)-2,-5,-7, ALP, COLL-1A2, OC, ON) in cells grown on the POL/nHA substrate. In addition, as compared with the POL surface, cells grown on the POL/nHA substrate demonstrated better osteoconductive properties, as demonstrated by the increase in adhesion and spreading, likely as a result of the increased surface roughness of the composite. Conclusions The increased expression of BMPs and osteoinductive biomarkers suggest that nano-hydroxyapatite may stimulate the proliferation and differentiation of local alveolar osteoblasts and thus encourage bone regeneration at sites of alveolar bone regeneration. PMID:24650194

  9. Machilin A isolated from Myristica fragrans stimulates osteoblast differentiation.

    PubMed

    Lee, Su-Ui; Shim, Ki Shuk; Ryu, Shi Yong; Min, Yong Ki; Kim, Seong Hwan

    2009-02-01

    This study evaluated the stimulatory effects of machilin A and structurally related lignans isolated from Myristica fragrans on osteoblast differentiation. In two IN VITRO osteoblast differentiation models, machilin A stimulated osteoblast differentiation via activation of p38 MAP kinase. Lignans isolated from Myristica fragrans also stimulated osteoblast differentiation in MC3T3-E1 cells; the lignans included macelignan, machilin F, nectandrin B, safrole, licarin A, licarin B, myristargenol, and meso-dihydroguaiaretic acid. These data suggest that lignans isolated from Myristica fragrans have anabolic activity in bone metabolism.

  10. Serotonin regulates osteoblast proliferation and function in vitro.

    PubMed

    Dai, S Q; Yu, L P; Shi, X; Wu, H; Shao, P; Yin, G Y; Wei, Y Z

    2014-09-01

    The monoamine serotonin (5-hydroxytryptamine, 5-HT), a well-known neurotransmitter, also has important functions outside the central nervous system. The objective of this study was to investigate the role of 5-HT in the proliferation, differentiation, and function of osteoblasts in vitro. We treated rat primary calvarial osteoblasts with various concentrations of 5-HT (1 nM to 10 µM) and assessed the rate of osteoblast proliferation, expression levels of osteoblast-specific proteins and genes, and the ability to form mineralized nodules. Next, we detected which 5-HT receptor subtypes were expressed in rat osteoblasts at different stages of osteoblast differentiation. We found that 5-HT could inhibit osteoblast proliferation, differentiation, and mineralization at low concentrations, but this inhibitory effect was mitigated at relatively high concentrations. Six of the 5-HT receptor subtypes (5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, and 5-HT2C) were found to exist in rat osteoblasts. Of these, 5-HT2A and 5-HT1B receptors had the highest expression levels, at both early and late stages of differentiation. Our results indicated that 5-HT can regulate osteoblast proliferation and function in vitro.

  11. Effects of Staphylococcus epidermidis on osteoblast cell adhesion and viability on a Ti alloy surface in a microfluidic co-culture environment.

    PubMed

    Lee, Joung-Hyun; Wang, Hongjun; Kaplan, Jeffrey B; Lee, Woo Y

    2010-11-01

    A microfluidic device was used for real time imaging of MC3T3-E1 murine calvarial pre-osteoblasts (osteoblasts) in response to very small numbers of Staphylococcus epidermidis inoculated on the surface of a polished TiAl6V4 alloy in a serum-based medium. The Ti alloy surface was integrated to a poly(dimethylsiloxane) fluidic housing with eight 10 μl channels for high-throughput, cross-contamination-free co-culture. In the absence of S. epidermidis osteoblasts were able to adhere, spread, proliferate and remain viable on the Ti alloy surface during a 25 h culture period. With 10(2) or 10(5) colony forming units (cfu) ml(-1) S. epidermidis inoculated on the alloy surface osteoblast adhesion, spreading and proliferation were not adversely affected during the early stages of culture. However, osteoblasts became damaged by the end of culture, as S. epidermidis actively proliferated in the co-culture channels and formed small clusters on the alloy surface. These observations suggest that the small numbers of S. epidermidis did not necessarily compete with osteoblasts for the alloy surface during initial host cell development, but rapid proliferation of the bacteria might have changed the microenvironment, making it unfavorable to sustain the viability of osteoblasts. The results provide a new insight in projecting the potential utility of the microfluidic co-culture approach to developing physiologically and clinically relevant in vitro models of orthopedic implant-associated bacterial infection.

  12. Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells

    SciTech Connect

    Levesque, Martine; Martineau, Corine; Jumarie, Catherine; Moreau, Robert

    2008-09-15

    Since bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation, alterations in osteoblast proliferation and differentiation may disturb the equilibrium of bone remodeling. Exposure to cadmium (Cd) has been associated with the alteration of bone metabolism and the development of osteoporosis. Because little information is available about the direct effects of Cd on osteoblastic cells, we have characterized in vitro the cellular accumulation and cytotoxicity of Cd in human osteoblastic cells. Incubation of osteoblast-like MG-63 cells with increasing concentrations of Cd in serum-free culture medium reduced cell viability in a time- and concentration-dependent manner, suggesting that Cd accumulates in osteoblasts. Consequently, an uptake time-course could be characterized for the cellular accumulation of {sup 109}Cd in serum-free culture medium. In order to characterize the mechanisms of Cd uptake, experiments have been conducted under well-defined metal speciation conditions in chloride and nitrate transport media. The results revealed a preferential uptake of Cd{sup 2+} species. The cellular accumulation and cytotoxicity of Cd increased in the absence of extracellular calcium (Ca), suggesting that Cd may enter the cells in part through Ca channels. However, neither the cellular accumulation nor the cytotoxicity of Cd was modified by voltage-dependent Ca channel (VDCC) modulators or potassium-induced depolarization. Moreover, exposure conditions activating or inhibiting capacitative Ca entry (CCE) failed to modify the cellular accumulation and cytotoxicity of Cd, which excludes the involvement of canonical transient receptor potential (TRPC) channels. The cellular accumulation and cytotoxicity of Cd were reduced by 2-APB, a known inhibitor of the Mg and Ca channel TRPM7 and were increased in the absence of extracellular magnesium (Mg). The inhibition of Cd uptake by Mg and Ca was not additive, suggesting

  13. Expression of thymosin beta-4 in human periodontal ligament cells and mouse periodontal tissue and its role in osteoblastic/cementoblastic differentiation.

    PubMed

    Lee, Sang-Im; Lee, Deok-Won; Yun, Hyung-Mun; Cha, Hee-Jae; Bae, Cheol-Hyeon; Cho, Eui-Sic; Kim, Eun-Cheol

    2015-01-01

    A recent report showed that thymosin beta-4 (Tβ4) is expressed during the development of tooth germ, but its effect on osteoblastic/cementoblastic differentiation is a controversial topic. Furthermore, the precise expression and function of Tβ4 in periodontal tissue remains unclear. Therefore, the purpose of this study was to investigate the immunolocalization of Tβ4 in the developing periodontium of mouse, the function of Tβ4 in osteoblastic/cementoblastic differentiation, and the underlying mechanism regulating periodontal regeneration in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. Tβ4 expression was observed in differentiating hPDLCs, osteoblasts of the periodontium during development, as well as in mature tissue. Higher Tβ4 expression was observed in hPDLCs than in cementoblasts and osteoblasts in the developing periodontium. The expression of Tβ4 mRNA and protein gradually increased during PDL cell differentiation. The downregulation of Tβ4 expression by Tβ4 siRNA transfection inhibited osteoblastic differentiation by decreasing calcium nodule formation, alkaline phosphatase (ALP) activity, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. In contrast, Tβ4 activation using a Tβ4 peptide, promoted these processes by activation of Akt, p38, ERK MAPKs, and the NF-κB pathway. The expression of nuclear NFATc1 was upregulated by Tβ4 peptide in hPDLCs. Inhibition of the calcineurin/NFATc1 pathway by cyclosporin A and FK506, attenuated Tβ4-induced osteoblastic differentiation and activation of Wnt-related genes, as well as nuclear β-catenin in hPDLCs. In conclusion, this study demonstrates, for the first time, that Tβ4 is expressed in developing periodontal tissue and that its expression is associated with osteoblastic/cementoblastic differentiation. These results suggests that Tβ4 is a potential therapeutic target for periodontal regeneration or bone disease. PMID:26361868

  14. Ectopic expression of SOX9 in osteoblasts alters bone mechanical properties.

    PubMed

    Liang, Bojian; Cotter, Meghan M; Chen, Dongxing; Hernandez, Christopher J; Zhou, Guang

    2012-02-01

    Osteoporosis is a common skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. We previously demonstrated that Col1a1-SOX9 transgenic (TG) mice, in which SOX9 specifically expresses in osteoblasts driven by a 2.3-kb Col1a1 promoter, display osteopenia during the early postnatal stage. In this study, to further analyze the osteopenia phenotype and especially the effect of the osteoblast-specific expression of SOX9 on bone mechanical properties, we performed bone geometry and mechanical property analysis of long bones from Col1a1-SOX9 TG mice and wild-type littermates (WT) at different time points. Interestingly, after body weight adjustment, TG mice had similar whole-bone strength as WT mice but significantly thinner cortical bone, lower elastic modulus, and higher moment of inertia. Thus, osteoblast-specific SOX9 expression results in altered bone structure and material properties. Furthermore, the expression levels of Pcna, Col1a1, osteocalcin, and the Opg/Rankl ratio in TG mice were significantly lower until 4 months of age compared with WT mice, suggesting that TG mice have dysregulated bone homeostasis. Finally, bone marrow stromal cells (MSCs) isolated from TG mice display enhanced adipocyte differentiation and decreased osteoblast differentiation in vitro, suggesting that osteoblast-specific expression of SOX9 can lead to altered mesenchymal stem cell differentiation potentials. In conclusion, our study implies that SOX9 activity has to be tightly regulated in the adult skeleton to ensure optimal bone quality. PMID:22143895

  15. Enhanced and suppressed mineralization by acetoacetate and β-hydroxybutyrate in osteoblast cultures.

    PubMed

    Saito, Akihiro; Yoshimura, Kentaro; Miyamoto, Yoichi; Kaneko, Kotaro; Chikazu, Daichi; Yamamoto, Matsuo; Kamijo, Ryutaro

    2016-04-29

    It is known that diabetes aggravates alveolar bone loss associated with periodontitis. While insulin depletion increases the blood concentration of ketone bodies, i.e., acetoacetate and β-hydroxybutyrate, their roles in bone metabolism have not been much studied until today. We investigated the effects of acetoacetate and β-hydroxybutyrate on mineralization of extracellular matrix in cultures of mouse osteoblastic MC3T3-E1 cells and primary mouse osteoblasts in the presence and absence of bone morphogenetic protein-2. Acetoacetate potentiated alkaline phosphatase activity in MC3T3-E1 cells in a concentration-dependent manner, ranging from physiological to pathological concentrations (0.05-5 mmol/L). In contrast, β-hydroxybutyrate lowered it in the same experimental settings. Mineralization in cultures of these cells was also up-regulated by acetoacetate and down-regulated by β-hydroxybutyrate. Similar results were obtained in cultures of mouse primary osteoblasts. Neither alkaline phosphatase mRNA nor its protein expression in MC3T3-E1 cells was affected by acetoacetate or β-hydroxybutyrate, indicating that these ketone bodies control the enzyme activity of alkaline phosphatase in osteoblasts and hence their mineralization bi-directionally. Finally, either gene silencing of monocarboxylate transporter-1, a major transmembrate transporter for ketone bodies, nullified the effects of ketone bodies on alkaline phosphatase activity in MC3T3-E1 cells. Collectively, we found that ketone bodies bidirectionally modulates osteoblast functions, which suggests that ketone bodies are important endogenous factors that regulate bone metabolism in both physiological and pathological situations. PMID:27018251

  16. Plasma Membrane Factor XIIIA Transglutaminase Activity Regulates Osteoblast Matrix Secretion and Deposition by Affecting Microtubule Dynamics

    PubMed Central

    Al-Jallad, Hadil F.; Myneni, Vamsee D.; Piercy-Kotb, Sarah A.; Chabot, Nicolas; Mulani, Amina; Keillor, Jeffrey W.; Kaartinen, Mari T.

    2011-01-01

    Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to ‘block –and-track’ enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics. PMID:21283799

  17. The effect of acoustic radiation force on osteoblasts in cell/hydrogel constructs for bone repair

    PubMed Central

    Veronick, James; Assanah, Fayekah; Nair, Lakshmi S; Vyas, Varun; Huey, Bryan

    2016-01-01

    Ultrasound, or the application of acoustic energy, is a minimally invasive technique that has been used in diagnostic, surgical, imaging, and therapeutic applications. Low-intensity pulsed ultrasound (LIPUS) has been used to accelerate bone fracture repair and to heal non-union defects. While shown to be effective the precise mechanism behind its utility is still poorly understood. In this study, we considered the possibility that LIPUS may be providing a physical stimulus to cells within bony defects. We have also evaluated ultrasound as a means of producing a transdermal physical force that could stimulate osteoblasts that had been encapsulated within collagen hydrogels and delivered to bony defects. Here we show that ultrasound does indeed produce a measurable physical force and when applied to hydrogels causes their deformation, more so as ultrasound intensity was increased or hydrogel stiffness decreased. MC3T3 mouse osteoblast cells were then encapsulated within hydrogels to measure the response to this force. Statistically significant elevated gene expression for alkaline phosphatase and osteocalcin, both well-established markers of osteoblast differentiation, was noted in encapsulated osteoblasts (p < 0.05), suggesting that the physical force provided by ultrasound may induce bone formation in part through physically stimulating cells. We have also shown that this osteoblastic response is dependent in part on the stiffness of the encapsulating hydrogel, as stiffer hydrogels resulted in reducing or reversing this response. Taken together this approach, encapsulating cells for implantation into a bony defect that can potentially be transdermally loaded using ultrasound presents a novel regenerative engineering approach to enhanced fracture repair. PMID:27229906

  18. Enhanced and suppressed mineralization by acetoacetate and β-hydroxybutyrate in osteoblast cultures.

    PubMed

    Saito, Akihiro; Yoshimura, Kentaro; Miyamoto, Yoichi; Kaneko, Kotaro; Chikazu, Daichi; Yamamoto, Matsuo; Kamijo, Ryutaro

    2016-04-29

    It is known that diabetes aggravates alveolar bone loss associated with periodontitis. While insulin depletion increases the blood concentration of ketone bodies, i.e., acetoacetate and β-hydroxybutyrate, their roles in bone metabolism have not been much studied until today. We investigated the effects of acetoacetate and β-hydroxybutyrate on mineralization of extracellular matrix in cultures of mouse osteoblastic MC3T3-E1 cells and primary mouse osteoblasts in the presence and absence of bone morphogenetic protein-2. Acetoacetate potentiated alkaline phosphatase activity in MC3T3-E1 cells in a concentration-dependent manner, ranging from physiological to pathological concentrations (0.05-5 mmol/L). In contrast, β-hydroxybutyrate lowered it in the same experimental settings. Mineralization in cultures of these cells was also up-regulated by acetoacetate and down-regulated by β-hydroxybutyrate. Similar results were obtained in cultures of mouse primary osteoblasts. Neither alkaline phosphatase mRNA nor its protein expression in MC3T3-E1 cells was affected by acetoacetate or β-hydroxybutyrate, indicating that these ketone bodies control the enzyme activity of alkaline phosphatase in osteoblasts and hence their mineralization bi-directionally. Finally, either gene silencing of monocarboxylate transporter-1, a major transmembrate transporter for ketone bodies, nullified the effects of ketone bodies on alkaline phosphatase activity in MC3T3-E1 cells. Collectively, we found that ketone bodies bidirectionally modulates osteoblast functions, which suggests that ketone bodies are important endogenous factors that regulate bone metabolism in both physiological and pathological situations.

  19. Cobalt and chromium exposure affects osteoblast function and impairs the mineralization of prosthesis surfaces in vitro.

    PubMed

    Shah, Karan M; Wilkinson, Jeremy Mark; Gartland, Alison

    2015-11-01

    Cobalt (Co) and chromium (Cr) ions and nanoparticles equivalent to those released through tribo-corrosion of prosthetic metal-on-metal (MOM) bearings and taper junctions are detrimental to osteoblast activity and function in vitro when examined as individual species. Here we examined the effects of Co(2+):Cr(3+) and Co(2+):Cr(6+) combinations on osteoblast-like SaOS-2 cellular activity, alkaline phosphatase (ALP) activity and mineralization to better reflect clinical exposure conditions in vivo. We also assessed the effect of Co(2+):Cr(3+) combinations and Co:Cr nanoparticles on SaOS-2 cell osteogenic responses on grit-blasted, plasma-sprayed titanium-coated, and hydroxyapatite-coated prosthesis surfaces. Cellular activity and ALP activity were reduced to a greater extent with combination treatments compared to individual ions. Co(2+) and Cr(3+) interacted additively and synergistically to reduce cellular activity and ALP activity, respectively, while the Co(2+) with Cr(6+) combination was dominated by the effect of Cr(6+) alone. Mineralization by osteoblasts was greater on hydroxyapatite-coated surfaces compared to grit-blasted and plasma-sprayed titanium-coated surfaces. Treatments with Co(2+):Cr(3+) ions and Co:Cr nanoparticles reduced the percentage mineralization on all surfaces, with hydroxyapatite-coated surfaces having the least reduction. In conclusion, our data suggests that previous studies investigating individual metal ions underestimate their potential clinical effects on osteoblast activity. Furthermore, the data suggests that hydroxyapatite-coated surfaces may modulate osteoblast responses to metal debris.

  20. Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation

    SciTech Connect

    Nifuji, Akira; Ideno, Hisashi; Ohyama, Yoshio; Takanabe, Rieko; Araki, Ryoko; Abe, Masumi; Noda, Masaki; Shibuya, Hiroshi

    2010-04-15

    Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLK in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.

  1. Signaling and transcriptional regulation in osteoblast commitment and differentiation

    PubMed Central

    Huang, Wei; Yang, Shuying; Shao, Jianzhong; Li, Yi-Ping

    2013-01-01

    The major event that triggers osteogenesis is the transition of mesenchymal stem cells into bone forming, differentiating osteoblast cells. Osteoblast differentiation is the primary component of bone formation, exemplified by the synthesis, deposition and mineralization of extracellular matrix. Although not well understood, osteoblast differentiation from mesenchymal stem cells is a well-orchestrated process. Recent advances in molecular and genetic studies using gene targeting in mouse enable a better understanding of the multiple factors and signaling networks that control the differentiation process at a molecular level. Osteoblast commitment and differentiation are controlled by complex activities involving signal transduction and transcriptional regulation of gene expression. We review Wnt signaling pathway and Runx2 regulation network, which are critical for osteoblast differentiation. Many other factors and signaling pathways have been implicated in regulation of osteoblast differentiation in a network manner, such as the factors Osterix, ATF4, and SATB2 and the TGF-beta, Hedgehog, FGF, ephrin, and sympathetic signaling pathways. This review summarizes the recent advances in the studies of signaling transduction pathways and transcriptional regulation of osteoblast cell lineage commitment and differentiation. The knowledge of osteoblast commitment and differentiation should be applied towards the development of new diagnostic and therapeutic alternatives for human bone diseases. PMID:17485283

  2. Aurantio-obtusin stimulates chemotactic migration and differentiation of MC3T3-E1 osteoblast cells.

    PubMed

    Vishnuprasad, Chethala N; Tsuchiya, Tomoko; Kanegasaki, Shiro; Kim, Joon Ho; Han, Sung Soo

    2014-05-01

    Osteoporosis is one of the major metabolic bone diseases and is among the most challenging noncommunicable diseases to treat. Although there is an increasing interest in identifying bioactive molecules for the prevention and management of osteoporosis, such studies principally focus only on differentiation and mineralization of osteoblasts or inhibition of osteoclast activity. Stimulation of osteoblast migration must be a promising osteoanabolic strategy for improved metabolic bone disease therapy. In this study, we show that an anthraquinone derivative, aurantio-obtusin, stimulated chemotactic migration of MC3T3-E1 osteoblast cells in a concentration-dependent manner. The use of a real-time chemotaxis analyzing system, TAXIScan, facilitated the evaluation of both velocity and directionality of osteoblast migration in response to the compound. Besides migration, the compound stimulated osteoblast differentiation and mineralization. Taken together, the data presented in this paper demonstrate that aurantio-obtusin is a promising osteoanabolic compound of natural origin with potential therapeutic applications in the prevention of osteoporosis and other metabolic bone diseases.

  3. Response of Osteoblasts to the Stimulus of Fluid Flow.

    PubMed

    Huang, Ling-Wei; Ren, Li; Yang, Peng-Fei; Shang, Peng

    2015-01-01

    Bone is an important porous tissue that supports the body, maintains calcium and phosphate homeostasis, protects vital organs, and houses bone marrow. The interaction between hydrostatic pressure and fluid phase, solid phase, cells, and vascular in bone makes bone inevitably bear baseline levels of fluid flow. Fluid flow plays an important role in regulating the proliferation, differentiation, distribution, and apoptosis of osteoblasts in bone. The effect of fluid flow on osteoblasts is dependent on time, velocity, and type. Some response of osteoblasts to fluid flow is closely related to the soluble factors secreted by the osteoblasts themselves or other types of bone cells. When the response is disordered, related bone diseases such as osteoporosis, osteoarthritis, and abnormal osteogenesis probably happen. In this article we review the current progress in the study of the response of osteoblasts to the direct and indirect stimulus of fluid flow and their roles in osteogenesis and related bone diseases.

  4. [Compatibility of a novel ethylenediamine modified polylactic acid with osteoblasts].

    PubMed

    Su, Aihua; Wang, Yuanliang; Luo, Yanfeng; Wu, Keda

    2005-08-01

    Biocompatibility of a newly developed ethylenediamine modified poly (DL-latic acid) (EMPLA) with osteoblasts was investigated by means of cell morphology and cell proliferation. Films of PLA and EMPLA were made by solvent casting. Osteoblasts obtained from crania of neonatal Wistar rats were cultured on surfaces of PLA and EMPLA, with glass as control. The cell morphology was observed by phase contrast microscope and the cell proliferation was determined by MTT assay. The morphology observations revealed that the osteoblasts cultured on EMPLA spread wider than those on PLA, and much more cells were confluent on EMPLA, compared to those on PLA and glass. The growth curves showed the osteoblasts on EMPLA grew faster than did those on PLA and glass. The results exhibited that the biocompatibility of EMPLA with osteoblasts is better than that of PLA and glass, which suggested wide applications of EMPLA in biomedical area, especially in tissue engineering. PMID:16156255

  5. Osteoblast mineralization with composite nanofibrous substrate for bone tissue regeneration.

    PubMed

    Venugopal, Jayarama Reddy; Giri Dev, Venkateshwarapuram Rengaswami; Senthilram, Thinakaran; Sathiskumar, Dhayalan; Gupta, Deepika; Ramakrishna, Seeram

    2011-01-01

    Several studies are currently ongoing to construct synthetic bone-like materials with composites of natural and polymeric materials with HA (hydroxyapatite). The present study aims to fabricate composite nanofibrous substrate of Chit/HA (chitosan/HA - 80:25) prepared by dissolving in TFA/DCM (trifluoroacetic acid/dichloromethane) (70:30, w/w) for 5 days and electrospun to fabricate a scaffold for bone tissue engineering. HA (25 wt %) was sonicated for 30 min to obtain a homogenous dispersion of nanoparticles within the Chit (80 wt %) matrix for fabricating composite nanofibrous scaffold (Chit/HA). The nanofibres of Chit and Chit/HA were obtained with fibre diameters of 274 ± 75 and 510 ± 198 nm, respectively, and characterized by FESEM (field emission scanning electron microscopy) and FTIR (Fourier transform infrared). The interaction of hFOBs (human fetal osteoblasts) and nanofibrous substrates were analysed for cell morphology (FESEM), mineralization [ARS (Alizarin Red-S) staining], quantification of minerals and finally identified the elements present in Chit/HA/osteoblasts by EDX (energy-dispersive X-ray) analysis. EDX analysis confirmed that the spherulites contain calcium and phosphorus, the major constituents in calcium phosphate apatite, the mineral phase of the bone. Mineralization was increased significantly (P<0.001) up to 108% in Chit/HA compared with Chit nanofibres. These results confirmed that the electrospun composite Chit/HA nanofibrous substrate is a potential biocomposite material for the proliferation and mineralization of hFOBs required for enhanced bone tissue regeneration. PMID:20923413

  6. Exosomes derived from mineralizing osteoblasts promote ST2 cell osteogenic differentiation by alteration of microRNA expression.

    PubMed

    Cui, Yazhou; Luan, Jing; Li, Haiying; Zhou, Xiaoyan; Han, Jinxiang

    2016-01-01

    Mineralizing osteoblasts (MOBs) can release exosomes, although the functional significance remains unclear. In the present study, we demonstrate that exosomes derived from mineralizing pre-osteoblast MC3T3-E1 cells can promote bone marrow stromal cell (ST2) differentiation to osteoblasts. We reveal that MOB-derived exosomes significantly influence miRNA profiles in recipient ST2 cells, and these changes tend to activate the Wnt signaling pathway by inhibiting Axin1 expression and increasing β-catenin expression. We also suggest that MOB derived-exosomes partly induce the variation in miRNA expression in recipient ST2 cells by exosomal miRNA transfer. These findings suggest an exosome-mediated mode of cell-to-cell communication in the osteogenic microenvironment, and also indicate the potential of MOB exosomes in bone tissue engineering.

  7. Hydrocarbon Deposition Attenuates Osteoblast Activity on Titanium

    PubMed Central

    Hayashi, R.; Ueno, T.; Migita, S.; Tsutsumi, Y.; Doi, H.; Ogawa, T.; Hanawa, T.; Wakabayashi, N.

    2014-01-01

    Although the reported percentage of bone-implant contact is far lower than 100%, the cause of such low levels of bone formation has rarely been investigated. This study tested the negative biological effect of hydrocarbon deposition onto titanium surfaces, which has been reported to be inevitable. Osteogenic MC3T3-E1 cells were cultured on titanium disks on which the carbon concentration was experimentally regulated to achieve carbon/titanium (C/Ti) ratios of 0.3, 0.7, and 1.0. Initial cellular activities such as cell attachment and cell spreading were concentration-dependently suppressed by the amount of carbon on the titanium surface. The osteoblastic functions of alkaline phosphatase activity and calcium mineralization were also reduced by more than 40% on the C/Ti (1.0) surface. These results indicate that osteoblast activity is influenced by the degree of hydrocarbon contamination on titanium implants and suggest that hydrocarbon decomposition before implant placement may increase the biocompatibility of titanium. PMID:24868012

  8. The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

    PubMed Central

    2010-01-01

    Background The proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined. Methods We evaluated the effects of dasatinib on bone marrow-derived mesenchymal stromal cells (MSC) differentiation into osteoblasts, in the presence or absence of a mixture of dexamethasone, ascorbic acid and β-glycerophosphate (DAG) for up to 21 days. The differentiation kinetics was assessed by evaluating mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers (receptor activator of nuclear factor kappa B ligand [RANKL], bone sialoprotein [BSP], osteopontin [OPN]). Results Dasatinib significantly increased the activity of ALP and the level of calcium deposition in MSC cultured with DAG after, respectively, 7 and 14 days; it upregulated the expression of BSP and OPN genes independently of DAG; and it markedly downregulated the expression of RANKL gene and protein (decrease in RANKL/OPG ratio), the key factor that stimulates osteoclast differentiation and activity. Conclusions Our results suggest a dual role for dasatinib in both (i) stimulating osteoblast differentiation leading to a direct increase in bone formation, and (ii) downregulating RANKL synthesis by osteoblasts leading to an indirect inhibition of osteoclastogenesis. Thus, dasatinib is a potentially interesting candidate drug for the treatment of osteolysis through its dual effect on bone metabolism. PMID:20565769

  9. Brucella abortus Invasion of Osteoblasts Inhibits Bone Formation

    PubMed Central

    Scian, Romina; Barrionuevo, Paula; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2012-01-01

    Osteoarticular brucellosis is the most common presentation of the active disease in humans. Loss of bone is a serious complication of localized bacterial infection of bones or the adjacent tissue, and brucellosis proved not to be the exception. The skeleton is a dynamic organ system which is constantly remodeled. Osteoblasts are responsible for the deposition of bone matrix and are thought to facilitate the calcification and mineralization of the bone matrix, and their function could be altered under infectious conditions. In this article, we describe immune mechanisms whereby Brucella abortus may invade and replicate within osteoblasts, inducing apoptosis, inhibiting mineral and organic matrix deposition, and inducing upregulation of RANKL expression. Additionally, all of these mechanisms contributed in different ways to bone loss. These processes implicate the activation of signaling pathways (mitogen-activated protein kinases [MAPK] and caspases) involved in cytokine secretion, expression of activating molecules, and cell death of osteoblasts. In addition, considering the relevance of macrophages in intracellular Brucella survival and proinflammatory cytokine secretion in response to infection, we also investigated the role of these cells as modulators of osteoblast survival, differentiation, and function. We demonstrated that supernatants from B. abortus-infected macrophages may also mediate osteoblast apoptosis and inhibit osteoblast function in a process that is dependent on the presence of tumor necrosis factor alpha (TNF-α). These results indicate that B. abortus may directly and indirectly harm osteoblast function, contributing to the bone and joint destruction observed in patients with osteoarticular complications of brucellosis. PMID:22547546

  10. Fibronectin is a survival factor for differentiated osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Doty, S. B.; Lull, J. C.; Holmuhamedov, E.; Humphries, M. J.; Damsky, C. H.

    1998-01-01

    The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin, which regulates the adhesion, differentiation and function of various adherent cells. Interactions with fibronectin are required for osteoblast differentiation in vitro, since fibronectin antagonists added to cultures of immature fetal calvarial osteoblasts inhibit their progressive differentiation. To determine if fibronectin plays a unique role in fully differentiated osteoblasts, cultures that had already formed mineralized nodules in vitro were treated with fibronectin antagonists. Fibronectin antibodies caused >95% of the cells in the mature cultures to display characteristic features of apoptosis (nuclear condensation, apoptotic body formation, DNA laddering) within 24 hours. Cells appeared to acquire sensitivity to fibronectin antibody-induced apoptosis as a consequence of differentiation, since antibodies failed to kill immature cells and the first cells killed were those associated with mature nodules. Intact plasma fibronectin, as well as fragments corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of fibronectin, independently induced apoptosis of mature (day-13), but not immature (day-4), osteoblasts. Finally, transforming growth factor-beta1 partially protected cells from the apoptotic effects of fibronectin antagonists. Thus, in the course of maturation cultured osteoblasts switch from depending on fibronectin for differentiation to depending on fibronectin for survival. These data suggest that fibronectin, together with transforming growth factor-beta1, may affect bone formation, in part by regulating the survival of osteoblasts.

  11. Effect of magnesium ion on human osteoblast activity

    PubMed Central

    He, L.Y.; Zhang, X.M.; Liu, B.; Tian, Y.; Ma, W.H.

    2016-01-01

    Magnesium, a promising biodegradable metal, has been reported in several studies to increase bone formation. Although there is some information regarding the concentrations of magnesium ions that affect bone remodeling at a cellular level, little is known about the effect of magnesium ions on cell gap junctions. Therefore, this study aimed to systematically investigate the effects of different concentrations of magnesium on bone cells, and further evaluate its effect on gap junctions of osteoblasts. Cultures of normal human osteoblasts were treated with magnesium ions at concentrations of 1, 2 and 3 mM, for 24, 48 and 72 h. The effects of magnesium ions on viability and function of normal human osteoblasts and on gap junction intercellular communication (GJIC) in osteoblasts were investigated. Magnesium ions induced significant (P<0.05) increases in cell viability, alkaline phosphate activity and osteocalcin levels of human osteoblasts. These stimulatory actions were positively associated with the concentration of magnesium and the time of exposure. Furthermore, the GJIC of osteoblasts was significantly promoted by magnesium ions. In conclusion, this study demonstrated that magnesium ions induced the activity of osteoblasts by enhancing GJIC between cells, and influenced bone formation. These findings may contribute to a better understanding of the influence of magnesium on bone remodeling and to the advance of its application in clinical practice. PMID:27383121

  12. Calcineurin/NFAT signaling in osteoblasts regulates bone mass.

    PubMed

    Winslow, Monte M; Pan, Minggui; Starbuck, Michael; Gallo, Elena M; Deng, Lei; Karsenty, Gerard; Crabtree, Gerald R

    2006-06-01

    Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts. PMID:16740479

  13. HDAC inhibition amplifies gap junction communication in neural progenitors: Potential for cell-mediated enzyme prodrug therapy

    SciTech Connect

    Khan, Zahidul . E-mail: Zahidul.Khan@ki.se; Akhtar, Monira; Asklund, Thomas; Juliusson, Bengt . E-mail: Tomas.Ekstrom@ki.se

    2007-08-01

    Enzyme prodrug therapy using neural progenitor cells (NPCs) as delivery vehicles has been applied in animal models of gliomas and relies on gap junction communication (GJC) between delivery and target cells. This study investigated the effects of histone deacetylase (HDAC) inhibitors on GJC for the purpose of facilitating transfer of therapeutic molecules from recombinant NPCs. We studied a novel immortalized midbrain cell line, NGC-407 of embryonic human origin having neural precursor characteristics, as a potential delivery vehicle. The expression of gap junction protein connexin 43 (C x 43) was analyzed by western blot and immunocytochemistry. While C x 43 levels were decreased in untreated differentiating NGC-407 cells, the HDAC inhibitor 4-phenylbutyrate (4-PB) increased C x 43 expression along with increased membranous deposition in both proliferating and differentiating cells. Simultaneously, Ser 279/282-phosphorylated form of C x 43 was declined in both culture conditions by 4-PB. The 4-PB effect in NGC-407 cells was verified by using HNSC.100 human neural progenitors and Trichostatin A. Improved functional GJC is of imperative importance for therapeutic strategies involving intercellular transport of low molecular-weight compounds. We show here an enhancement by 4-PB, of the functional GJC among NGC-407 cells, as well as between NGC-407 and human glioma cells, as indicated by increased fluorescent dye transfer.

  14. Targeting the osteoblast: approved and experimental anabolic agents for the treatment of osteoporosis.

    PubMed

    Toulis, Konstantinos A; Anastasilakis, Athanasios D; Polyzos, Stergios A; Makras, Polyzois

    2011-01-01

    Targeting osteoblast may be the means of effectively improving both bone quality and mass, thus offering an intriguing alternative in the treatment of osteoporosis. Aside from injectable parathyroid hormone (PTH) and its novel preparations, PTH-related peptide (PTHrP), calcilytics, beta-adrenergic receptors, enhancement of Wnt signaling (mainly via sclerostin and Dickkopf-1 neutralization), regulation of low-density lipoprotein receptor-related protein (LPR) 5/osteoblast axis, activin, IGF-1, and bone morphogenic proteins (BMPs) are reviewed for their basic rationale and evidence of bone anabolic potential. Sclerostin neutralizing antibody, teriparatide transdermal patch, and PTHrP (1-36) are currently at an advanced stage of research. Safety and tissue specificity are the prerequisites in the development of a novel treatment, especially when addressing a chronic condition such as osteoporosis.

  15. Pinctada fucata mantle gene 4 (PFMG4) from pearl oyster mantle enhances osteoblast differentiation.

    PubMed

    Wang, Xiaoyan; Harimoto, Kenichi; Fuji, Ryosuke; Liu, Jing; Li, Liyuan; Wang, Pan; Akaike, Toshihiro; Wang, Zhao

    2015-01-01

    The organic matrix of nacre has been reported for its effect on osteogenesis. It was found that PFMG4 (Pinctada fucata mantle gene 4) with an N-terminal signal peptide could be secreted into nacre of Pinctada fucata (P. fucata). Here, we report that PFMG4 is highly expressed in mantle tissue and has high homology with C1q protein in different species. In MC3T3-E1 osteoblast cells, we found that highly expressed PFMG4 could suppress cell proliferation and type I collagen expression, but it could increase alkaline phosphatase activity and mineralized deposition. These results show that PFMG4 has potential ability in enhancing osteoblast differentiation, suggesting a new idea in developing medicine for the therapy of osteoporosis.

  16. Use of Primary Calvarial Osteoblasts to Evaluate the Function of Wnt Signaling in Osteogenesis.

    PubMed

    Zhong, Zhendong A; Ethen, Nicole J; Williams, Bart O

    2016-01-01

    In vitro culture and genetic manipulation of primary calvarial cell cultures is a convenient and robust system to investigate gene function in osteoblast differentiation. We have used this system to study the functions of many genes in the Wnt signaling pathway within osteoblasts. Here, we describe a detailed protocol outlining the establishment and characterization of primary calvarial cells from mice carrying a conditionally inactivatable allele of the Wntless (Wls) gene (Wls (flox/flox)). We previously used this approach to delete the Wntless gene by infecting with a Cre-expressing adenovirus, and to evaluate the effects of Wnt signaling loss on osteogenic potential in osteogenic medium with ascorbic acid. This detailed protocol is adaptable to use with any floxed allele. PMID:27590158

  17. The Effect of Electric and Magnetic Fields on Protein Self-Organization and Osteoblast Biomineralization

    NASA Astrophysics Data System (ADS)

    Ba, Xiaolan; Fourman, Lara; Singal, Sanchita; Meng, Yizhi; Rafailovich, Miriam

    2009-03-01

    The induction of bone formation to an intentional orientation is a potentially viable clinical treatment for bone regeneration. Among the many chemical and physical factors, electric and magnetic fields are an essential way to regulate the behavior of cells and matrix fibers. The aims of this study are to investigate the effects of electric and magnetic fields on protein self-organization and osteoblast biomineralization on polymer surfaces in vitro. To this end, we use atomic force microscopy (AFM) to characterize the morphology of protein fiber and ECM by cells. The mechanical property of protein fibers was investigated by shear modulation force microscopy (SMFM). The late-stage of mineralization was characterized by scanning electron microscopy (SEM) and grazing incident x-ray diffraction (GIXD). The primary data indicated that the magnetic field could enhance the biomineralization of osteoblast.

  18. Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis

    PubMed Central

    ABUNA, Rodrigo Paolo Flores; STRINGHETTA-GARCIA, Camila Tami; FIORI, Leonardo Pimentel; DORNELLES, Rita Cassia Menegati; ROSA, Adalberto Luiz; BELOTI, Marcio Mateus

    2016-01-01

    ABSTRACT Aging negatively affects bone/titanium implant interactions. Our hypothesis is that the unbalance between osteogenesis and adipogenesis induced by aging may be involved in this phenomenon. Objective We investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) from young and aged rats cultured on Ti. Material and Methods Bone marrow MSCs derived from 1-month and 21-month rats were cultured on Ti discs under osteogenic conditions for periods of up to 21 days and osteoblast and adipocyte markers were evaluated. Results Cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of RUNX2, osterix, ALP, bone sialoprotein, osteopontin, and osteocalcin were reduced in cultures of 21-month rats compared with 1-month rats grown on Ti. Gene expression of PPAR-γ , adipocyte protein 2, and resistin and lipid accumulation were increased in cultures of 21-month rats compared with 1-month rats grown on the same conditions. Conclusions These results indicate that the lower osteogenic potential of MSCs derived from aged rats compared with young rats goes along with the higher adipogenic potential in cultures grown on Ti surface. This unbalance between osteoblast and adipocyte differentiation should be considered in dental implant therapy to the elderly population. PMID:27556209

  19. Abdominal Fat and Sarcopenia in Women Significantly Alter Osteoblasts Homeostasis In Vitro by a WNT/β-Catenin Dependent Mechanism

    PubMed Central

    Wannenes, Francesca; Papa, Vincenza; Greco, Emanuela A.; Fornari, Rachele; Marocco, Chiara; Di Luigi, Luigi; Donini, Lorenzo M.; Lenzi, Andrea

    2014-01-01

    Obesity and sarcopenia have been associated with mineral metabolism derangement and low bone mineral density (BMD). We investigated whether imbalance of serum factors in obese or obese sarcopenic patients could affect bone cell activity in vitro. To evaluate and characterize potential cellular and molecular changes of human osteoblasts, cells were exposed to sera of four groups of patients: (1) affected by obesity with normal BMD (O), (2) affected by obesity with low BMD (OO), (3) affected by obesity and sarcopenia (OS), and (4) affected by obesity, sarcopenia, and low BMD (OOS) as compared to subjects with normal body weight and normal BMD (CTL). Patients were previously investigated and characterized for body composition, biochemical and bone turnover markers. Then, sera of different groups of patients were used to incubate human osteoblasts and evaluate potential alterations in cell homeostasis. Exposure to OO, OS, and OOS sera significantly reduced alkaline phosphatase, osteopontin, and BMP4 expression compared to cells exposed to O and CTL, indicating a detrimental effect on osteoblast differentiation. Interestingly, sera of all groups of patients induced intracellular alteration in Wnt/β-catenin molecular pathway, as demonstrated by the significant alteration of specific target genes expression and by altered β-catenin cellular compartmentalization and GSK3β phosphorylation. In conclusion our results show for the first time that sera of obese subjects with low bone mineral density and sarcopenia significantly alter osteoblasts homeostasis in vitro, indicating potential detrimental effects of trunk fat on bone formation and skeletal homeostasis. PMID:24963291

  20. Abdominal Fat and Sarcopenia in Women Significantly Alter Osteoblasts Homeostasis In Vitro by a WNT/ β -Catenin Dependent Mechanism.

    PubMed

    Wannenes, Francesca; Papa, Vincenza; Greco, Emanuela A; Fornari, Rachele; Marocco, Chiara; Baldari, Carlo; Di Luigi, Luigi; Emerenziani, Gian Pietro; Poggiogalle, Eleonora; Guidetti, Laura; Donini, Lorenzo M; Lenzi, Andrea; Migliaccio, Silvia

    2014-01-01

    Obesity and sarcopenia have been associated with mineral metabolism derangement and low bone mineral density (BMD). We investigated whether imbalance of serum factors in obese or obese sarcopenic patients could affect bone cell activity in vitro. To evaluate and characterize potential cellular and molecular changes of human osteoblasts, cells were exposed to sera of four groups of patients: (1) affected by obesity with normal BMD (O), (2) affected by obesity with low BMD (OO), (3) affected by obesity and sarcopenia (OS), and (4) affected by obesity, sarcopenia, and low BMD (OOS) as compared to subjects with normal body weight and normal BMD (CTL). Patients were previously investigated and characterized for body composition, biochemical and bone turnover markers. Then, sera of different groups of patients were used to incubate human osteoblasts and evaluate potential alterations in cell homeostasis. Exposure to OO, OS, and OOS sera significantly reduced alkaline phosphatase, osteopontin, and BMP4 expression compared to cells exposed to O and CTL, indicating a detrimental effect on osteoblast differentiation. Interestingly, sera of all groups of patients induced intracellular alteration in Wnt/ β -catenin molecular pathway, as demonstrated by the significant alteration of specific target genes expression and by altered β -catenin cellular compartmentalization and GSK3 β phosphorylation. In conclusion our results show for the first time that sera of obese subjects with low bone mineral density and sarcopenia significantly alter osteoblasts homeostasis in vitro, indicating potential detrimental effects of trunk fat on bone formation and skeletal homeostasis. PMID:24963291

  1. The influence of MicroRNA-150 in Osteoblast Matrix Mineralization.

    PubMed

    Dong, Chun-Ling; Liu, Hao-Zhi; Zhang, Zhen-Chun; Zhao, Huan-Li; Zhao, Hui; Huang, Yan; Yao, Jian-Hua; Sun, Tian-Sheng

    2015-12-01

    This study investigated the influence of miR-150 expression on osteoblast matrix mineralization and its mechanisms. The mouse osteoblast cell line MC3T3-E1 was used as an in vitro model of bone formation. On the fifth day of mineralization, transfection experiments using agomiR-150, agomiR-NC, antagomiR-150 antagomiR-NC, and mock groups were set up to test the effects of miR-150 in MC3T3-E1 model. The mRNA and protein levels of OC, ALP, type I collagen, and OPN were measured by qRT-PCR and ELISA. Matrix mineralization was detected by alizarin red S (ARS) staining and flow cytometry was employed to quantify apoptosis in each group. RT-PCR and Western blot were applied to detect the expression of target gene MMP14. Our results demonstrated that the endogenous expression levels of miR-150, OC, ALP, type I collagen, and OPN in MC3T3-E1 cells increased steadily. Exogenous expressions of agomiR-150 and antagomiR-150 can significantly up-/down-regulate, respectively, the expression level of miR-150 in MC3T3-E1 cells. Compared with the mock group, higher expression levels of OC, ALP, type I collagen, and OPN mRNA were observed in the agomiR-150 group, while lower mRNA expression levels of OC, ALP, type I collagen, and OPN were found in the antagomiR-150 group. Based on these results, potential miR-150 targeted genes are discussed. Our results showed that miR-150 supports the osteoblastic phenotype related to osteoblast function and bone mineralization. Thus, miR-150 may have potential therapeutic applications in promoting bone formation in certain disease settings, such as in osteoporosis and in elderly patients.

  2. miRNA-132-3p inhibits osteoblast differentiation by targeting Ep300 in simulated microgravity

    PubMed Central

    Hu, Zebing; Wang, Yixuan; Sun, Zhongyang; Wang, Han; Zhou, Hua; Zhang, Lianchang; Zhang, Shu; Cao, Xinsheng

    2015-01-01

    Recent studies have demonstrated that miRNAs can play important roles in osteoblast differentiation and bone formation. However, the function of miRNAs in bone loss induced by microgravity remains unclear. In this study, we investigated the differentially expressed miRNAs in both the femur tissues of hindlimb unloading rats and primary rat osteoblasts (prOB) exposed to simulated microgravity. Specifically, miR-132-3p was found up-regulated and negatively correlated with osteoblast differentiation. Overexpression of miR-132-3p significantly inhibited prOB differentiation, whereas inhibition of miR-132-3p function yielded an opposite effect. Furthermore, silencing of miR-132-3p expression effectively attenuated the negative effects of simulated microgravity on prOB differentiation. Further experiments confirmed that E1A binding protein p300 (Ep300), a type of histone acetyltransferase important for Runx2 activity and stability, was a direct target of miR-132-3p. Up-regulation of miR-132-3p by simulated microgravity could inhibit osteoblast differentiation in part by decreasing Ep300 protein expression, which, in turn, resulted in suppression of the activity and acetylation of Runx2, a key regulatory factor of osteoblast differentiation. Taken together, our findings are the first to demonstrate that miR-132-3p can inhibit osteoblast differentiation and participate in the regulation of bone loss induced by simulated microgravity, suggesting a potential target for counteracting decreases in bone formation. PMID:26686902

  3. Suppression of osteoblast differentiation during weightlessness

    NASA Technical Reports Server (NTRS)

    Roberts, W. E.; Mozsary, P. G.; Morey, E. R.

    1982-01-01

    It is pointed out that associated with weightlessness is a marked depression or arrest of bone formation. Although the mechanism of this effect is unknown, it probably involves a failure of osteogenic induction. The present study's objective is to determine if weightlessness alters osteoblast differentiation, as evidenced by a change in relative distribution of large to small nuclei in rat moral periodontal ligament of the maxilla. In conjunction with the U.S./USSR Biological Satellite Program, male Wistar rats were flown aboard a modified Soviet Vostok spacecraft (Cosmos 1129). The results of the study are discussed. Morphometric investigations suggest that depleted numbers of preosteoblasts may be an important factor in the inhibition of bone formation during weightlessness.

  4. Glimepiride Promotes Osteogenic Differentiation in Rat Osteoblasts via the PI3K/Akt/eNOS Pathway in a High Glucose Microenvironment

    PubMed Central

    Xiong, Wei; Tan, Baosheng; Geng, Wei; Li, Jun; Liu, Hongchen

    2014-01-01

    Our previous studies demonstrated that glimepiride enhanced the proliferation and differentiation of osteoblasts and led to activation of the PI3K/Akt pathway. Recent genetic evidence shows that endothelial nitric oxide synthase (eNOS) plays an important role in bone homeostasis. In this study, we further elucidated the roles of eNOS, PI3K and Akt in bone formation by osteoblasts induced by glimepiride in a high glucose microenvironment. We demonstrated that high glucose (16.5 mM) inhibits the osteogenic differentiation potential and proliferation of rat osteoblasts. Glimepiride activated eNOS expression in rat osteoblasts cultured with two different concentrations of glucose. High glucose-induced osteogenic differentiation was significantly enhanced by glimepiride. Down-regulation of PI3K P85 levels by treatment with LY294002 (a PI3K inhibitor) led to suppression of P-eNOS and P-AKT expression levels, which in turn resulted in inhibition of RUNX2, OCN and ALP mRNA expression in osteoblasts induced by glimepiride at both glucose concentrations. ALP activity was partially inhibited by 10 µM LY294002. Taken together, our results demonstrate that glimepiride-induced osteogenic differentiation of osteoblasts occurs via eNOS activation and is dependent on the PI3K/Akt signaling pathway in a high glucose microenvironment. PMID:25391146

  5. Glimepiride promotes osteogenic differentiation in rat osteoblasts via the PI3K/Akt/eNOS pathway in a high glucose microenvironment.

    PubMed

    Ma, Pan; Gu, Bin; Xiong, Wei; Tan, Baosheng; Geng, Wei; Li, Jun; Liu, Hongchen

    2014-01-01

    Our previous studies demonstrated that glimepiride enhanced the proliferation and differentiation of osteoblasts and led to activation of the PI3K/Akt pathway. Recent genetic evidence shows that endothelial nitric oxide synthase (eNOS) plays an important role in bone homeostasis. In this study, we further elucidated the roles of eNOS, PI3K and Akt in bone formation by osteoblasts induced by glimepiride in a high glucose microenvironment. We demonstrated that high glucose (16.5 mM) inhibits the osteogenic differentiation potential and proliferation of rat osteoblasts. Glimepiride activated eNOS expression in rat osteoblasts cultured with two different concentrations of glucose. High glucose-induced osteogenic differentiation was significantly enhanced by glimepiride. Down-regulation of PI3K P85 levels by treatment with LY294002 (a PI3K inhibitor) led to suppression of P-eNOS and P-AKT expression levels, which in turn resulted in inhibition of RUNX2, OCN and ALP mRNA expression in osteoblasts induced by glimepiride at both glucose concentrations. ALP activity was partially inhibited by 10 µM LY294002. Taken together, our results demonstrate that glimepiride-induced osteogenic differentiation of osteoblasts occurs via eNOS activation and is dependent on the PI3K/Akt signaling pathway in a high glucose microenvironment. PMID:25391146

  6. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels.

    PubMed

    Maes, Christa; Kobayashi, Tatsuya; Selig, Martin K; Torrekens, Sophie; Roth, Sanford I; Mackem, Susan; Carmeliet, Geert; Kronenberg, Henry M

    2010-08-17

    During endochondral bone development, the first osteoblasts differentiate in the perichondrium surrounding avascular cartilaginous rudiments; the source of trabecular osteoblasts inside the later bone is, however, unknown. Here, we generated tamoxifen-inducible transgenic mice bred to Rosa26R-LacZ reporter mice to follow the fates of stage-selective subsets of osteoblast lineage cells. Pulse-chase studies showed that osterix-expressing osteoblast precursors, labeled in the perichondrium prior to vascular invasion of the cartilage, give rise to trabecular osteoblasts, osteocytes, and stromal cells inside the developing bone. Throughout the translocation, some precursors were found to intimately associate with invading blood vessels, in pericyte-like fashion. A similar coinvasion occurs during endochondral healing of bone fractures. In contrast, perichondrial mature osteoblasts did not exhibit perivascular localization and remained in the outer cortex of developing bones. These findings reveal the specific involvement of immature osteoblast precursors in the coupled vascular and osteogenic transformation essential to endochondral bone development and repair.

  7. Proinflammatory Response of Human Osteoblastic Cell Lines and Osteoblast-Monocyte Interaction upon Infection with Brucella spp.▿

    PubMed Central

    Delpino, M. Victoria; Fossati, Carlos A.; Baldi, Pablo C.

    2009-01-01

    The ability of Brucella spp. to infect human osteoblasts and the cytokine response of these cells to infection were investigated in vitro. Brucella abortus, B. suis, B. melitensis, and B. canis were able to infect the SaOS-2 and MG-63 osteoblastic cell lines, and the first three species exhibited intracellular replication. B. abortus internalization was not significantly affected by pretreatment of cells with cytochalasin D but was inhibited up to 92% by colchicine. A virB10 mutant of B. abortus could infect but not replicate within osteoblasts, suggesting a role for the type IV secretion system in intracellular survival. Infected osteoblasts produced low levels of chemokines (interleukin-8 [IL-8] and macrophage chemoattractant protein 1 [MCP-1]) and did not produce proinflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor alpha [TNF-α]). However, osteoblasts stimulated with culture supernatants from Brucella-infected human monocytes (THP-1 cell line) produced chemokines at levels 12-fold (MCP-1) to 17-fold (IL-8) higher than those of infected osteoblasts and also produced IL-6. In the inverse experiment, culture supernatants from Brucella-infected osteoblasts induced the production of IL-8, IL-1β, IL-6, and TNF-α by THP-1 cells. The induction of TNF-α and IL-1β was largely due to granulocyte-macrophage colony-stimulating factor produced by infected osteoblasts, as demonstrated by inhibition with a specific neutralizing antibody. This study shows that Brucella can invade and replicate within human osteoblastic cell lines, which can directly and indirectly mount a proinflammatory response. Both phenomena may have a role in the chronic inflammation and bone and joint destruction observed in osteoarticular brucellosis. PMID:19103778

  8. Fibronectin and Cyclic Strain Improve Cardiac Progenitor Cell Regenerative Potential In Vitro.

    PubMed

    French, Kristin M; Maxwell, Joshua T; Bhutani, Srishti; Ghosh-Choudhary, Shohini; Fierro, Marcos J; Johnson, Todd D; Christman, Karen L; Taylor, W Robert; Davis, Michael E

    2016-01-01

    Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2-4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment. PMID:27610140

  9. Fibronectin and Cyclic Strain Improve Cardiac Progenitor Cell Regenerative Potential In Vitro

    PubMed Central

    Ghosh-Choudhary, Shohini; Fierro, Marcos J.; Christman, Karen L.; Taylor, W. Robert

    2016-01-01

    Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2–4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment. PMID:27610140

  10. Fibronectin and Cyclic Strain Improve Cardiac Progenitor Cell Regenerative Potential In Vitro

    PubMed Central

    Ghosh-Choudhary, Shohini; Fierro, Marcos J.; Christman, Karen L.; Taylor, W. Robert

    2016-01-01

    Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2–4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment.

  11. Connexins and pannexins in the skeleton: gap junctions, hemichannels and more

    PubMed Central

    Plotkin, Lilian I.; Stains, Joseph P.

    2015-01-01

    Regulation of bone homeostasis depends on the concerted actions of bone-forming osteoblasts and bone-resorbing osteoclasts, controlled by osteocytes, cells derived from osteoblasts surrounded by bone matrix. The control of differentiation, viability and function of bone cells relies on the presence of connexins. Connexin43 regulates the expression of genes required for osteoblast and osteoclast differentiation directly or by changing the levels of osteocytic genes, and connexin45 may oppose connexin43 actions in osteoblastic cells. Connexin37 is required for osteoclast differentiation and its deletion results in increased bone mass. Less is known on the role of connexins in cartilage, ligaments and tendons. Connexin43, connexin45, connexin32, connexin46 and connexin29 are expressed in chondrocytes, while connexin43 and connexin32 are expressed in ligaments and tendons. Similarly, although the expression of pannexin1, pannexin2 and pannexin3 has been demonstrated in bone and cartilage cells, their function in these tissues is not fully understood. PMID:26091748

  12. GATA4 negatively regulates bone sialoprotein expression in osteoblasts

    PubMed Central

    Song, Insun; Jeong, Byung-chul; Choi, Yong Jun; Chung, Yoon-Sok; Kim, Nacksung

    2016-01-01

    GATA4 has been reported to act as a negative regulator in osteoblast differentiation by inhibiting the Dlx5 transactivation of Runx2 via the attenuation of the binding ability of Dlx5 to the Runx2 promoter region. Here, we determine the role of GATA4 in the regulation of bone sialoprotein (Bsp) in osteoblasts. We observed that the overexpression of Runx2 or Sox9 induced the Bsp expression in osteoblastic cells. Silencing GATA4 further enhanced the Runx2- and Sox9-mediated Bsp promoter activity, whereas GATA4 overexpression down-regulated Bsp promoter activity mediated by Runx2 and Sox9. GATA4 also interacted with Runx2 and Sox9, by attenuating the binding ability of Runx2 and Sox9 to the Bsp promoter region. Our data suggest that GATA4 acts as a negative regulator of Bsp expression in osteoblasts. [BMB Reports 2016; 49(6): 343-348] PMID:26973342

  13. Role of Integrin in Mechanical Loading of Osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, Ruth; Demsky, Caroline

    2000-01-01

    Mechanical forces generated by gravity, weightbearing, and muscle contraction play a key role in the genesis and maintenance of skeletal structure. The molecular mechanisms that mediate changes in osteoblast activity in response to altered patterns of skeletal loading are not known, and a better understanding of these processes may be essential for developing effective treatment strategies to prevent disuse osteoporosis. We have elucidated specific integrin/ECM (extracellular matrix) interactions that are required for osteoblast differentiation and survival and have developed a useful loading system to further explore the molecular basis of mechano-sensitivity of osteoblasts. The long term goal of our collaborative research is to understand how the ECM and cell adhesion proteins and integrins interaction to mediate the response of osteoblasts and their progenitors to mechanical loading. We suggest that integrin/ECM interactions are crucial for basic cellular processes, including differentiation and survival, as well as to participate in detecting and mediating cellular responses to mechanical stimuli.

  14. Mechanisms regulating osteoblast response to surface microtopography and vitamin D

    NASA Astrophysics Data System (ADS)

    Bell, Bryan Frederick, Jr.

    A comprehensive understanding of the interactions between orthopaedic and dental implant surfaces with the surrounding host tissue is essential in the design of advanced biomaterials that better promote bone growth and osseointegration of implants. Dental implants with roughened surfaces and high surface energy are well known to promote osteoblast differentiation in vitro and promote increased bone-to-implant contact in vivo. In addition, increased surface roughness increases osteoblasts response to the vitamin D metabolite 1alpha,25(OH)2D3. However, the exact mechanisms mediating cell response to surface properties and 1alpha,25(OH)2D3 are still being elucidated. The central aim of the thesis is to investigate whether integrin signaling in response to rough surface microtopography enhances osteoblast differentiation and responsiveness to 1alpha,25(OH)2D3. The hypothesis is that the integrin alpha5beta1 plays a role in osteoblast response to surface microtopography and that 1alpha,25(OH) 2D3 acts through VDR-independent pathways involving caveolae to synergistically enhance osteoblast response to surface roughness and 1alpha,25(OH) 2D3. To test this hypothesis the objectives of the studies performed in this thesis were: (1) to determine if alpha5beta 1 signaling is required for osteoblast response to surface microstructure; (2) to determine if increased responsiveness to 1alpha,25(OH)2D 3 requires the vitamin D receptor, (3) to determine if rough titanium surfaces functionalized with the peptides targeting integrins (RGD) and transmembrane proteoglycans (KRSR) will enhance both osteoblast proliferation and differentiation, and (4) to determine whether caveolae, which are associated with integrin and 1alpha,25(OH)2D3 signaling, are required for enhance osteogenic response to surface microstructure and 1alpha,25(OH)2D 3. The results demonstrate that integrins, VDR, and caveolae play important roles in mediating osteoblast response to surface properties and 1alpha,25

  15. H19 activates Wnt signaling and promotes osteoblast differentiation by functioning as a competing endogenous RNA

    PubMed Central

    Liang, Wei-Cheng; Fu, Wei-Ming; Wang, Yu-Bing; Sun, Yu-Xin; Xu, Liang-Liang; Wong, Cheuk-Wa; Chan, Kai-Ming; Li, Gang; Waye, Mary Miu-Yee; Zhang, Jin-Fang

    2016-01-01

    Bone homeostasis is tightly orchestrated and maintained by the balance between osteoblasts and osteoclasts. Recent studies have greatly expanded our understanding of the molecular mechanisms of cellular differentiation. However, the functional roles of non-coding RNAs particularly lncRNAs in remodeling bone architecture remain elusive. In our study, lncRNA H19 was found to be upregulated during osteogenesis in hMSCs. Stable expression of H19 significantly accelerated in vivo and in vitro osteoblast differentiation. Meanwhile, by using bioinformatic investigations and RIP assays combined with luciferase reporter assays, we demonstrated that H19 functioned as an miRNA sponge for miR-141 and miR-22, both of which were negative regulators of osteogenesis and Wnt/β-catenin pathway. Further investigations revealed that H19 antagonized the functions of these two miRNAs and led to de-repression of their shared target gene β-catenin, which eventually activated Wnt/β-catenin pathway and hence potentiated osteogenesis. In addition, we also identified a novel regulatory feedback loop between H19 and its encoded miR-675-5p. And miR-675-5p was found to directly target H19 and counteracted osteoblast differentiation. To sum up, these observations indicate that the lncRNA H19 modulates Wnt/β-catenin pathway by acting as a competing endogenous RNA, which may shed light on the functional role of lncRNAs in coordinating osteogenesis. PMID:26853553

  16. Sulfated hyaluronan improves bone regeneration of diabetic rats by binding sclerostin and enhancing osteoblast function.

    PubMed

    Picke, Ann-Kristin; Salbach-Hirsch, Juliane; Hintze, Vera; Rother, Sandra; Rauner, Martina; Kascholke, Christian; Möller, Stephanie; Bernhardt, Ricardo; Rammelt, Stefan; Pisabarro, M Teresa; Ruiz-Gómez, Gloria; Schnabelrauch, Matthias; Schulz-Siegmund, Michaela; Hacker, Michael C; Scharnweber, Dieter; Hofbauer, Christine; Hofbauer, Lorenz C

    2016-07-01

    Bone fractures in patients with diabetes mellitus heal poorly and require innovative therapies to support bone regeneration. Here, we assessed whether sulfated hyaluronan included in collagen-based scaffold coatings can improve fracture healing in diabetic rats. Macroporous thermopolymerized lactide-based scaffolds were coated with collagen including non-sulfated or sulfated hyaluronan (HA/sHA3) and inserted into 3 mm femoral defects of non-diabetic and diabetic ZDF rats. After 12 weeks, scaffolds coated with collagen/HA or collagen/sHA3 accelerated bone defect regeneration in diabetic, but not in non-diabetic rats as compared to their non-coated controls. At the tissue level, collagen/sHA3 promoted bone mineralization and decreased the amount of non-mineralized bone matrix. Moreover, collagen/sHA3-coated scaffolds from diabetic rats bound more sclerostin in vivo than the respective controls. Binding assays confirmed a high binding affinity of sHA3 to sclerostin. In vitro, sHA3 induced BMP-2 and lowered the RANKL/OPG expression ratio, regardless of the glucose concentration in osteoblastic cells. Both sHA3 and high glucose concentrations decreased the differentiation of osteoclastic cells. In summary, scaffolds coated with collagen/sHA3 represent a potentially suitable biomaterial to improve bone defect regeneration in diabetic conditions. The underlying mechanism involves improved osteoblast function and binding sclerostin, a potent inhibitor of Wnt signaling and osteoblast function. PMID:27131598

  17. Electrospun Scaffolds for Osteoblast Cells: Peptide-Induced Concentration-Dependent Improvements of Polycaprolactone

    PubMed Central

    Dettin, Monica; Zamuner, Annj; Roso, Martina; Gloria, Antonio; Iucci, Giovanna; Messina, Grazia M. L.; D'Amora, Ugo; Marletta, Giovanni; Modesti, Michele; Castagliuolo, Ignazio; Brun, Paola

    2015-01-01

    The design of hybrid poly-ε-caprolactone (PCL)-self-assembling peptides (SAPs) matrices represents a simple method for the surface functionalization of synthetic scaffolds, which is essential for cell compatibility. This study investigates the influence of increasing concentrations (2.5%, 5%, 10% and 15% w/w SAP compared to PCL) of three different SAPs on the physico-chemical/mechanical and biological properties of PCL fibers. We demonstrated that physico-chemical surface characteristics were slightly improved at increasing SAP concentrations: the fiber diameter increased; surface wettability increased with the first SAP addition (2.5%) and slightly less for the following ones; SAP-surface density increased but no change in the conformation was registered. These results could allow engineering matrices with structural characteristics and desired wettability according to the needs and the cell system used. The biological and mechanical characteristics of these scaffolds showed a particular trend at increasing SAP concentrations suggesting a prevailing correlation between cell behavior and mechanical features of the matrices. As compared with bare PCL, SAP enrichment increased the number of metabolic active h-osteoblast cells, fostered the expression of specific osteoblast-related mRNA transcripts, and guided calcium deposition, revealing the potential application of PCL-SAP scaffolds for the maintenance of osteoblast phenotype. PMID:26361004

  18. Sulfated hyaluronan improves bone regeneration of diabetic rats by binding sclerostin and enhancing osteoblast function.

    PubMed

    Picke, Ann-Kristin; Salbach-Hirsch, Juliane; Hintze, Vera; Rother, Sandra; Rauner, Martina; Kascholke, Christian; Möller, Stephanie; Bernhardt, Ricardo; Rammelt, Stefan; Pisabarro, M Teresa; Ruiz-Gómez, Gloria; Schnabelrauch, Matthias; Schulz-Siegmund, Michaela; Hacker, Michael C; Scharnweber, Dieter; Hofbauer, Christine; Hofbauer, Lorenz C

    2016-07-01

    Bone fractures in patients with diabetes mellitus heal poorly and require innovative therapies to support bone regeneration. Here, we assessed whether sulfated hyaluronan included in collagen-based scaffold coatings can improve fracture healing in diabetic rats. Macroporous thermopolymerized lactide-based scaffolds were coated with collagen including non-sulfated or sulfated hyaluronan (HA/sHA3) and inserted into 3 mm femoral defects of non-diabetic and diabetic ZDF rats. After 12 weeks, scaffolds coated with collagen/HA or collagen/sHA3 accelerated bone defect regeneration in diabetic, but not in non-diabetic rats as compared to their non-coated controls. At the tissue level, collagen/sHA3 promoted bone mineralization and decreased the amount of non-mineralized bone matrix. Moreover, collagen/sHA3-coated scaffolds from diabetic rats bound more sclerostin in vivo than the respective controls. Binding assays confirmed a high binding affinity of sHA3 to sclerostin. In vitro, sHA3 induced BMP-2 and lowered the RANKL/OPG expression ratio, regardless of the glucose concentration in osteoblastic cells. Both sHA3 and high glucose concentrations decreased the differentiation of osteoclastic cells. In summary, scaffolds coated with collagen/sHA3 represent a potentially suitable biomaterial to improve bone defect regeneration in diabetic conditions. The underlying mechanism involves improved osteoblast function and binding sclerostin, a potent inhibitor of Wnt signaling and osteoblast function.

  19. Stretching-induced nanostructures on shape memory polyurethane films and their regulation to osteoblasts morphology.

    PubMed

    Xing, Juan; Ma, Yufei; Lin, Manping; Wang, Yuanliang; Pan, Haobo; Ruan, Changshun; Luo, Yanfeng

    2016-10-01

    Programming such as stretching, compression and bending is indispensible to endow polyurethanes with shape memory effects. Despite extensive investigations on the contributions of programming processes to the shape memory effects of polyurethane, less attention has been paid to the nanostructures of shape memory polyurethanes surface during the programming process. Here we found that stretching could induce the reassembly of hard domains and thereby change the nanostructures on the film surfaces with dependence on the stretching ratios (0%, 50%, 100%, and 200%). In as-cast polyurethane films, hard segments sequentially assembled into nano-scale hard domains, round or fibrillar islands, and fibrillar apophyses. Upon stretching, the islands packed along the stretching axis to form reoriented fibrillar apophyses along the stretching direction. Stretching only changed the chemical patterns on polyurethane films without significantly altering surface roughness, with the primary composition of fibrillar apophyses being hydrophilic hard domains. Further analysis of osteoblasts morphology revealed that the focal adhesion formation and osteoblasts orientation were in accordance with the chemical patterns of the underlying stretched films, which corroborates the vital roles of stretching-induced nanostructures in regulating osteoblasts morphology. These novel findings suggest that programming might hold great potential for patterning polyurethane surfaces so as to direct cellular behavior. In addition, this work lays groundwork for guiding the programming of shape memory polyurethanes to produce appropriate nanostructures for predetermined medical applications.

  20. Genetic effect of zirconium oxide coating on osteoblast-like cells.

    PubMed

    Sollazzo, Vincenzo; Palmieri, Annalisa; Pezzetti, Furio; Bignozzi, Carlo Alberto; Argazzi, Roberto; Massari, Leo; Brunelli, Giorgio; Carinci, Francesco

    2008-02-01

    Zirconium is widely used as material for prosthetic devices because its good mechanical and chemical properties. When exposed to oxygen, zirconium becomes zirconium oxide (ZrO(2)), which is biocompatible. ZrO(2) can be also prepared as a colloidal suspension and then used to coat surfaces. Zirconium oxide coating (ZrO(2)C) can potentially have specific biologic effects, and among them is bone formation related to implant osseointegration. How this biomaterial alters osteoblast activity to promote bone formation is poorly understood. We therefore attempted to address this question by using microarray techniques to identify genes that are differently regulated in osteoblasts exposed to ZrO(2)C. By using DNA microarrays containing 20,000 genes, we identified in osteoblast-like cell lines (MG-63) cultured with ZrO(2)C several genes whose expression was significantly upregulated or downregulated. The differentially expressed genes cover a broad range of functional activities: (a) cell cycle regulation, (b) signal transduction, (c) immunity, and (d) cytoskeleton component. The data reported are, to our knowledge, the first genetic portrait of ZrO(2)C effects. They can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

  1. Electrospun Scaffolds for Osteoblast Cells: Peptide-Induced Concentration-Dependent Improvements of Polycaprolactone.

    PubMed

    Dettin, Monica; Zamuner, Annj; Roso, Martina; Gloria, Antonio; Iucci, Giovanna; Messina, Grazia M L; D'Amora, Ugo; Marletta, Giovanni; Modesti, Michele; Castagliuolo, Ignazio; Brun, Paola

    2015-01-01

    The design of hybrid poly-ε-caprolactone (PCL)-self-assembling peptides (SAPs) matrices represents a simple method for the surface functionalization of synthetic scaffolds, which is essential for cell compatibility. This study investigates the influence of increasing concentrations (2.5%, 5%, 10% and 15% w/w SAP compared to PCL) of three different SAPs on the physico-chemical/mechanical and biological properties of PCL fibers. We demonstrated that physico-chemical surface characteristics were slightly improved at increasing SAP concentrations: the fiber diameter increased; surface wettability increased with the first SAP addition (2.5%) and slightly less for the following ones; SAP-surface density increased but no change in the conformation was registered. These results could allow engineering matrices with structural characteristics and desired wettability according to the needs and the cell system used. The biological and mechanical characteristics of these scaffolds showed a particular trend at increasing SAP concentrations suggesting a prevailing correlation between cell behavior and mechanical features of the matrices. As compared with bare PCL, SAP enrichment increased the number of metabolic active h-osteoblast cells, fostered the expression of specific osteoblast-related mRNA transcripts, and guided calcium deposition, revealing the potential application of PCL-SAP scaffolds for the maintenance of osteoblast phenotype. PMID:26361004

  2. Proliferation and differentiation of osteoblast-like cells on apatite-wollastonite/polyethylene composites.

    PubMed

    Rea, S M; Brooks, R A; Best, S M; Kokubo, T; Bonfield, W

    2004-08-01

    Glass-ceramic apatite-wollastonite (A-W)/high-density polyethylene composite (AWPEX) materials have been designed to match the mechanical strength of human cortical bone and to provide favourable bioactivity, with potential use in many orthopaedic applications. To better understand AWPEX properties, the effects of surface finish and ceramic filler size and content on osteoblast-like cell attachment, proliferation, and differentiation were examined. Glass-ceramic content was tested at 30 and 50 vol% and median particle size at 4.5 and 7.7 microm. Samples were prepared as 1 x 10 x 10 mm(3) tiles with polished or rough surfaces, sterilized by gamma irradiation (2.5 Mrad), and characterized by scanning electron microscopy (SEM) and surface profilometry. Saos-2 human osteoblast-like cells were cultured on each surface at an initial concentration of 4500 cells/cm(2) for 1, 3, or 7 days. At each time point, adenosine triphosphate and alkaline phosphatase levels were measured to assess cell number and osteoblast differentiation. SEM imaging of cells on the composite surfaces showed preferential cell attachment to filler particles within the polymer matrix. Significant biochemical assay differences were found at 7 days, confirmed by ANOVA post-hoc testing using Bonferroni's correction. Overall, increased exposure of the glass-ceramic A-W phase in AWPEX through surface polishing, higher volume fraction and/or larger particle size was found to lead to an improved cell response.

  3. Nck influences preosteoblastic/osteoblastic migration and bone mass

    PubMed Central

    Aryal A.C, Smriti; Miyai, Kentaro; Izu, Yayoi; Hayata, Tadayoshi; Notomi, Takuya; Noda, Masaki; Ezura, Yoichi

    2015-01-01

    Migration of the cells in osteoblastic lineage, including preosteoblasts and osteoblasts, has been postulated to influence bone formation. However, the molecular bases that link preosteoblastic/osteoblastic cell migration and bone formation are incompletely understood. Nck (noncatalytic region of tyrosine kinase; collectively referred to Nck1 and Nck2) is a member of the signaling adaptors that regulate cell migration and cytoskeletal structures, but its function in cells in the osteoblastic lineage is not known. Therefore, we examined the role of Nck in migration of these cells. Nck is expressed in preosteoblasts/osteoblasts, and its knockdown suppresses migration as well as cell spreading and attachment to substrates. In contrast, Nck1 overexpression enhances spreading and increases migration and attachment. As for signaling, Nck double knockdown suppresses migration toward IGF1 (insulin-like growth factor 1). In these cells, Nck1 binds to IRS-1 (insulin receptor substrate 1) based on immunoprecipitation experiments using anti-Nck and anti–IRS-1 antibodies. In vivo, Nck knockdown suppresses enlargement of the pellet of DiI-labeled preosteoblasts/osteoblasts placed in the calvarial defects. Genetic experiments indicate that conditional double deletion of both Nck1 and Nck2 specifically in osteoblasts causes osteopenia. In these mice, Nck double deficiency suppresses the levels of bone-formation parameters such as bone formation rate in vivo. Interestingly, bone-resorption parameters are not affected. Finally, Nck deficiency suppresses repair of bone injury after bone marrow ablation. These results reveal that Nck regulates preosteoblastic/osteoblastic migration and bone mass. PMID:26621720

  4. Nck influences preosteoblastic/osteoblastic migration and bone mass.

    PubMed

    Aryal A C, Smriti; Miyai, Kentaro; Izu, Yayoi; Hayata, Tadayoshi; Notomi, Takuya; Noda, Masaki; Ezura, Yoichi

    2015-12-15

    Migration of the cells in osteoblastic lineage, including preosteoblasts and osteoblasts, has been postulated to influence bone formation. However, the molecular bases that link preosteoblastic/osteoblastic cell migration and bone formation are incompletely understood. Nck (noncatalytic region of tyrosine kinase; collectively referred to Nck1 and Nck2) is a member of the signaling adaptors that regulate cell migration and cytoskeletal structures, but its function in cells in the osteoblastic lineage is not known. Therefore, we examined the role of Nck in migration of these cells. Nck is expressed in preosteoblasts/osteoblasts, and its knockdown suppresses migration as well as cell spreading and attachment to substrates. In contrast, Nck1 overexpression enhances spreading and increases migration and attachment. As for signaling, Nck double knockdown suppresses migration toward IGF1 (insulin-like growth factor 1). In these cells, Nck1 binds to IRS-1 (insulin receptor substrate 1) based on immunoprecipitation experiments using anti-Nck and anti-IRS-1 antibodies. In vivo, Nck knockdown suppresses enlargement of the pellet of DiI-labeled preosteoblasts/osteoblasts placed in the calvarial defects. Genetic experiments indicate that conditional double deletion of both Nck1 and Nck2 specifically in osteoblasts causes osteopenia. In these mice, Nck double deficiency suppresses the levels of bone-formation parameters such as bone formation rate in vivo. Interestingly, bone-resorption parameters are not affected. Finally, Nck deficiency suppresses repair of bone injury after bone marrow ablation. These results reveal that Nck regulates preosteoblastic/osteoblastic migration and bone mass.

  5. Periodontal Specific Differentiation of Dental Follicle Stem Cells into Osteoblast, Fibroblast, and Cementoblast.

    PubMed

    Sowmya, S; Chennazhi, K P; Arzate, Higinio; Jayachandran, P; Nair, Shantikumar V; Jayakumar, R

    2015-10-01

    The dental follicle is a source of dental follicle stem cells (DFCs), which have the potential to differentiate into the periodontal lineage. DFCs therefore are of value in dental tissue engineering. The purpose of this study was to evaluate the effect of growth factor type and concentration on DFC differentiation into periodontal specific lineages. DFCs were isolated from the human dental follicle and characterized for the expression of mesenchymal markers. The cells were positive for CD-73, CD-44, and CD-90; and negative for CD-33, CD-34, and CD-45. The expression of CD-29 and CD-31 was almost negligible. The cells also expressed periodontal ligament and cementum markers such as periodontal ligament-associated protein-1 (PLAP-1), fibroblast growth factor-2 (FGF-2), and cementum protein-1 (CEMP-1), however, the expression of osteoblast markers was absent. Further, the DFCs were cultured in three different induction medium to analyze the osteoblastic, fibroblastic, and cementoblastic differentiation. Runt-related transcription factor 2 (RUNX-2), alkaline phosphatase (ALP) activity, alizarin staining, calcium quantification, collagen type-1 (Col-1), and osteopontin (OPN) expression confirmed the osteoblastic differentiation of DFCs. DFCs cultured in recombinant human FGF-2 (rhFGF-2) containing medium showed enhanced PLAP-1, FGF-2, and COL-1 expression with increasing concentration of rhFGF-2 which thereby confirmed periodontal ligament fibroblastic differentiation. Similarly, DFCs cultured in recombinant human cementum protein-1 (rhCEMP-1) containing medium showed enhanced bone sialoprotein-2 (BSP-2), CEMP-1, and COL-1 expression with respect to rhCEMP-1 which confirmed cementoblastic differentiation. The expression of osteoblast, fibroblast, and cementoblast-related genes of DFCs cultured in induction medium was enhanced in comparison to DFCs cultured in noninduction medium. Thus, growth factor-dependent differentiation of DFCs into periodontal specific lineages

  6. Genome-Wide Chromatin Landscape Transitions Identify Novel Pathways in Early Commitment to Osteoblast Differentiation

    PubMed Central

    Thompson, Bethtrice; Varticovski, Lyuba; Baek, Songjoon; Hager, Gordon L.

    2016-01-01

    Bone continuously undergoes remodeling by a tightly regulated process that involves osteoblast differentiation from Mesenchymal Stem Cells (MSC). However, commitment of MSC to osteoblastic lineage is a poorly understood process. Chromatin organization functions as a molecular gatekeeper of DNA functions. Detection of sites that are hypersensitive to Dnase I has been used for detailed examination of changes in response to hormones and differentiation cues. To investigate the early steps in commitment of MSC to osteoblasts, we used a model human temperature-sensitive cell line, hFOB. When shifted to non-permissive temperature, these cells undergo "spontaneous" differentiation that takes several weeks, a process that is greatly accelerated by osteogenic induction media. We performed Dnase I hypersensitivity assays combined with deep sequencing to identify genome-wide potential regulatory events in cells undergoing early steps of commitment to osteoblasts. Massive reorganization of chromatin occurred within hours of differentiation. Whereas ~30% of unique DHS sites were located in the promoters, the majority was outside of the promoters, designated as enhancers. Many of them were at novel genomic sites and need to be confirmed experimentally. We developed a novel method for identification of cellular networks based solely on DHS enhancers signature correlated to gene expression. The analysis of enhancers that were unique to differentiating cells led to identification of bone developmental program encompassing 147 genes that directly or indirectly participate in osteogenesis. Identification of these pathways provided an unprecedented view of genomic regulation during early steps of differentiation and changes related to WNT, AP-1 and other pathways may have therapeutic implications. PMID:26890492

  7. Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro

    SciTech Connect

    Aisha, M.D.; Nor-Ashikin, M.N.K.; Sharaniza, A.B.R.; Nawawi, H.; Froemming, G.R.A.

    2015-09-10

    Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases. - Highlights: • OFS stress transmits anabolic signals to osteoblasts. • Actin and tubulin fibers are rearranged under OFS stress. • OFS stress increases mitochondrial metabolism and proliferation. • Reduced RANKL/OPG ratio in response to OFS inhibits osteoclastogenesis. • OFS stress prevents apoptosis and stimulates ALP and OCN.

  8. The effects of bone pâté on human osteoblasts cell cultures.

    PubMed

    Quaranta, Nicola; Buccoliero, Cinzia; De Luca, Concetta; Mori, Giorgio; Brunetti, Giacomina; Colucci, Silvia; Colaianni, Graziana; Grano, Maria

    2016-06-01

    The aim of the present study was to evaluate the effect of bone pate on human osteoblast differentiation by measuring cell viability, alkaline phosphatase activity and expression of the transcription factors and of the major components of the extracellular matrix. Although bone paté has been used in ear surgery for many years and when placed in contact with mastoid and external auditory canal bone become viable, the cellular mechanisms that lead to its osteointegration have never been described. Bone paté taken from four patients subjected to mastoidectomy and affected by middle ear and mastoid cholesteatoma was placed in contact with osteoblast-like cell cultures. Four experimental conditions were obtained: cell cultures treated with bone patè, with bone paté mixed with fibrin glue, with fibrin glue and untreated. After 24 h, the viability of the cells was evaluated; after 1 week, alkaline phosphatase activity and the expression of transcription factors and bone matrix proteins were assessed by quantitative polymerase chain reaction. After 24 h osteoblasts showed increased viability when treated with bone paté (19 % increase) and bone pate mixed with fibrin glue (34 % increase). After 1 week, the number of alkaline phosphatase positive cells increased by 97 and 94 % in cultures treated with bone paté alone and bone pate mixed with fibrin glue. Treatment with bone patè upregulated transcription factors and components of the extracellular matrix. The present data show that bone paté has a high osteoinductive potential on human osteoblasts, enhancing their activity. PMID:26133919

  9. Novel osteoblast-adhesive peptides for dental/orthopedic biomaterials.

    PubMed

    Dettin, Monica; Conconi, Maria Teresa; Gambaretto, Roberta; Pasquato, Antonella; Folin, Marcella; Di Bello, Carlo; Parnigotto, Pier Paolo

    2002-06-01

    Next generation dental/orthopedic biomaterials must be designed to enhance and support osteoblast adhesion. The osteoblasts use different ways to adhere, that is, integrin- and proteoglycan-mediated mechanisms. The present study reports on the synthesis and osteoblast-adhesive properties of peptides carrying RGD motifs and of sequences mapped on human vitronectin. Our data suggest that osteoblast adhesion on polystyrene plates modified with a linear peptide, in which the GRGDSP sequence is repeated four times, was significantly higher when compared to the adhesion obtained using branched peptides, interestingly containing the same motif. Osteoblast adhesion assays on acellular bone matrix using this active peptide gave very promising results. We also demonstrated that a novel peptide, carrying the X-B-B-B-X-B-B-X motif (where B is a basic amino acid and X is a nonbasic residue), promotes proteoglycan-mediated osteoblast adhesion more efficiently with respect to the KRSR sequence that was recently proposed as heparan-sulfate binding peptide. PMID:11920671

  10. Time-lapse Raman imaging of osteoblast differentiation

    NASA Astrophysics Data System (ADS)

    Hashimoto, Aya; Yamaguchi, Yoshinori; Chiu, Liang-Da; Morimoto, Chiaki; Fujita, Katsumasa; Takedachi, Masahide; Kawata, Satoshi; Murakami, Shinya; Tamiya, Eiichi

    2015-07-01

    Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.

  11. Candida glabrata survives and replicates in human osteoblasts.

    PubMed

    Muñoz-Duarte, Ana Rosa; Castrejón-Jiménez, Nayeli Shantal; Baltierra-Uribe, Shantal Lizbeth; Pérez-Rangel, Sofia Judith; Carapia-Minero, Natalee; Castañeda-Sánchez, Jorge Ismael; Luna-Herrera, Julieta; López-Santiago, Rubén; Rodríguez-Tovar, Aída Verónica; García-Pérez, Blanca Estela

    2016-06-01

    Candida glabrata is an opportunistic pathogen that is considered the second most common cause of candidiasis after Candida albicans Many characteristics of its mechanisms of pathogenicity remain unknown. Recent studies have focused on determining the events that underlie interactions between C. glabrata and immune cells, but the relationship between this yeast and osteoblasts has not been studied in detail. The aim of this study was to determine the mechanisms of interaction between human osteoblasts and C. glabrata, and to identify the roles played by some of the molecules that are produced by these cells in response to infection. We show that C. glabrata adheres to and is internalized by human osteoblasts. Adhesion is independent of opsonization, and internalization depends on the rearrangement of the actin cytoskeleton. We show that C. glabrata survives and replicates in osteoblasts and that this intracellular behavior is related to the level of production of nitric oxide and reactive oxygen species. Opsonized C. glabrata stimulates the production of IL-6, IL-8 and MCP-1 cytokines. Adhesion and internalization of the pathogen and the innate immune response of osteoblasts require viable C. glabrata These results suggest that C. glabrata modulates immunological mechanisms in osteoblasts to survive inside the cell.

  12. Time-lapse Raman imaging of osteoblast differentiation

    PubMed Central

    Hashimoto, Aya; Yamaguchi, Yoshinori; Chiu, Liang-da; Morimoto, Chiaki; Fujita, Katsumasa; Takedachi, Masahide; Kawata, Satoshi; Murakami, Shinya; Tamiya, Eiichi

    2015-01-01

    Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable. PMID:26211729

  13. Increased osteoblast adhesion on nanograined Ti modified with KRSR.

    PubMed

    Balasundaram, Ganesan; Webster, Thomas J

    2007-03-01

    Peptide sequences such as lysine-arginine-serine-arginine (KRSR) selectively bind transmembrane proteoglycans (e.g. heparin sulfate) of osteoblasts (bone-forming cells) and are, therefore, actively being investigated for orthopedic applications. Further, nanophase materials (or materials with grain or particle sizes less than 100 nm) are promising new materials that promote new bone growth more than compared to conventional (that is, micron grain or particle size) materials. To combine the above two promising approaches for improving orthopedic implants, the objective of this in vitro study was to functionalize titanium (Ti) surfaces (both nanophase and conventional) with KRSR peptides and study their osteoblast cell adhesive properties. Materials were characterized by X-ray photoelectron spectroscopy, scanning electron microscopy, and atomic force microscopy. Results of this in vitro study provided evidence of increased osteoblast adhesion on nanophase compared to conventional Ti whether functionalized with KRSR or not. Results further showed that the immobilization of KRSR onto Ti (both nanophase and conventional) increased osteoblast adhesion compared to respective nonfunctionalized Ti and those functionalized with the negative control peptide KSRR. Most importantly, osteoblast adhesion on nonfunctionalized nanophase Ti increased compared to conventional Ti functionalized with KRSR. Further, select initial osteoblast adhesion was observed to occur at particle boundaries for any type of nanophase and conventional Ti formulated in this study. In summary, results provided evidence that not only should nonfunctionalized nanophase Ti be further studied for improved orthopedic applications but so should nanophase Ti functionalized with KRSR.

  14. mTORC1 Signaling Promotes Osteoblast Differentiation from Preosteoblasts

    PubMed Central

    Chen, Jianquan; Long, Fanxin

    2015-01-01

    Preosteoblasts are precursor cells that are committed to the osteoblast lineage. Differentiation of these cells to mature osteoblasts is regulated by the extracellular factors and environmental cues. Recent studies have implicated mTOR signaling in the regulation of osteoblast differentiation. However, mTOR exists in two distinct protein complexes (mTORC1 and mTORC2), and the specific role of mTORC1 in regulating the progression of preosteoblasts to mature osteoblastis still unclear. In this study, we first deleted Raptor, a unique and essential component of mTORC1, in primary calvarial cells. Deletion of Raptor resulted in loss of mTORC1 but an increase in mTORC2 signaling without overtly affecting autophagy. Under the osteogenic culture condition, Raptor-deficient cells exhibited a decrease in matrix synthesis and mineralization. qPCR analyses revealed that deletion of Raptor reduced the expression of late-stage markers for osteoblast differentiation (Bglap, Ibsp, and Col1a), while slightly increasing early osteoblast markers (Runx2, Sp7, and Alpl). Consistent with the findings in vitro, genetic ablation of Raptor in osterix-expressing cells led to osteopenia in mice. Together, our findings have identified a specific role for mTORC1 in the transition from preosteoblasts to mature osteoblasts. PMID:26090674

  15. Regulation of Osteoblast Survival by the Extracellular Matrix and Gravity

    NASA Technical Reports Server (NTRS)

    Globus. Ruth K.; Almeida, Eduardo A. C.; Searby, Nancy D.; Bowley, Susan M. (Technical Monitor)

    2000-01-01

    Spaceflight adversely affects the skeleton, posing a substantial risk to astronaut's health during long duration missions. The reduced bone mass observed in growing animals following spaceflight is due at least in part to inadequate bone formation by osteoblasts. Thus, it is of central importance to identify basic cellular mechanisms underlying normal bone formation. The fundamental ideas underlying our research are that interactions between extracellular matrix proteins, integrin adhesion receptors, cytoplasmic signaling and cytoskeletal proteins are key ingredients for the proper functioning of osteoblasts, and that gravity impacts these interactions. As an in vitro model system we used primary fetal rat calvarial cells which faithfully recapitulate osteoblast differentiation characteristically observed in vivo. We showed that specific integrin receptors ((alpha)3(beta)1), ((alpha)5(beta)1), ((alpha)8(betal)1) and extracellular matrix proteins (fibronectin, laminin) were needed for the differentiation of immature osteoblasts. In the course of maturation, cultured osteoblasts switched from depending on fibronectin and laminin for differentiation to depending on these proteins for their very survival. Furthermore, we found that manipulating the gravity vector using ground-based models resulted in activation of key intracellular survival signals generated by integrin/extracellular matrix interactions. We are currently testing the in vivo relevance of some of these observations using targeted transgenic technology. In conclusion, mechanical factors including gravity may participate in regulating survival via cellular interactions with the extracellular matrix. This leads us to speculate that microgravity adversely affects the survival of osteoblasts and contributes to spaceflight-induced osteoporosis.

  16. Candida glabrata survives and replicates in human osteoblasts.

    PubMed

    Muñoz-Duarte, Ana Rosa; Castrejón-Jiménez, Nayeli Shantal; Baltierra-Uribe, Shantal Lizbeth; Pérez-Rangel, Sofia Judith; Carapia-Minero, Natalee; Castañeda-Sánchez, Jorge Ismael; Luna-Herrera, Julieta; López-Santiago, Rubén; Rodríguez-Tovar, Aída Verónica; García-Pérez, Blanca Estela

    2016-06-01

    Candida glabrata is an opportunistic pathogen that is considered the second most common cause of candidiasis after Candida albicans Many characteristics of its mechanisms of pathogenicity remain unknown. Recent studies have focused on determining the events that underlie interactions between C. glabrata and immune cells, but the relationship between this yeast and osteoblasts has not been studied in detail. The aim of this study was to determine the mechanisms of interaction between human osteoblasts and C. glabrata, and to identify the roles played by some of the molecules that are produced by these cells in response to infection. We show that C. glabrata adheres to and is internalized by human osteoblasts. Adhesion is independent of opsonization, and internalization depends on the rearrangement of the actin cytoskeleton. We show that C. glabrata survives and replicates in osteoblasts and that this intracellular behavior is related to the level of production of nitric oxide and reactive oxygen species. Opsonized C. glabrata stimulates the production of IL-6, IL-8 and MCP-1 cytokines. Adhesion and internalization of the pathogen and the innate immune response of osteoblasts require viable C. glabrata These results suggest that C. glabrata modulates immunological mechanisms in osteoblasts to survive inside the cell. PMID:27073253

  17. Evolution of the osteoblast: skeletogenesis in gar and zebrafish

    PubMed Central

    2012-01-01

    Background Although the vertebrate skeleton arose in the sea 500 million years ago, our understanding of the molecular fingerprints of chondrocytes and osteoblasts may be biased because it is informed mainly by research on land animals. In fact, the molecular fingerprint of teleost osteoblasts differs in key ways from that of tetrapods, but we do not know the origin of these novel gene functions. They either arose as neofunctionalization events after the teleost genome duplication (TGD), or they represent preserved ancestral functions that pre-date the TGD. Here, we provide evolutionary perspective to the molecular fingerprints of skeletal cells and assess the role of genome duplication in generating novel gene functions. We compared the molecular fingerprints of skeletogenic cells in two ray-finned fish: zebrafish (Danio rerio)--a teleost--and the spotted gar (Lepisosteus oculatus)--a "living fossil" representative of a lineage that diverged from the teleost lineage prior to the TGD (i.e., the teleost sister group). We analyzed developing embryos for expression of the structural collagen genes col1a2, col2a1, col10a1, and col11a2 in well-formed cartilage and bone, and studied expression of skeletal regulators, including the transcription factor genes sox9 and runx2, during mesenchymal condensation. Results Results provided no evidence for the evolution of novel functions among gene duplicates in zebrafish compared to the gar outgroup, but our findings shed light on the evolution of the osteoblast. Zebrafish and gar chondrocytes both expressed col10a1 as they matured, but both species' osteoblasts also expressed col10a1, which tetrapod osteoblasts do not express. This novel finding, along with sox9 and col2a1 expression in developing osteoblasts of both zebrafish and gar, demonstrates that osteoblasts of both a teleost and a basally diverging ray-fin fish express components of the supposed chondrocyte molecular fingerprint. Conclusions Our surprising finding that

  18. Role of connexin 43 in vascular hyperpermeability and relationship to Rock1-MLC20 pathway in septic rats.

    PubMed

    Zhang, Jie; Yang, Guang-Ming; Zhu, Yu; Peng, Xiao-Yong; Li, Tao; Liu, Liang-Ming

    2015-12-01

    Connexin (Cx)43 has been shown to participate in several cardiovascular diseases. Increased vascular permeability is a common and severe complication in sepsis or septic shock. Whether or not Cx43 takes part in the regulation of vascular permeability in severe sepsis is not known, and the underlying mechanism has not been described. With cecal ligation and puncture-induced sepsis in rats and lipopolysaccharide (LPS)-treated vascular endothelial cells (VECs) from pulmonary veins, the role of Cx43 in increased vascular permeability and its relationship to the RhoA/Rock1 pathway were studied. It was shown that vascular permeability in the lungs, kidneys, and mesentery in sepsis rats and LPS-stimulated monolayer pulmonary vein VECs was significantly increased and positively correlated with the increased expression of Cx43 and Rock1 in these organs and cultured pulmonary vein VECs. The connexin inhibitor carbenoxolone (10 mg/kg iv) and the Rock1 inhibitor Y-27632 (2 mg/kg iv) alleviated the vascular leakage of lung, mesentery, and kidney in sepsis rats. Overexpressed Cx43 increased the phosphorylation of 20-kDa myosin light chain (MLC20) and the expression of Rock1 and increased the vascular permeability and decreased the transendothelial electrical resistance of pulmonary vein VECs. Cx43 RNA interference decreased the phosphorylation of MLC20 and the expression of Rock1 and decreased LPS-stimulated hyperpermeability of cultured pulmonary vein VECs. The Rock1 inhibitor Y-27632 alleviated LPS- and overexpressed Cx43-induced hyperpermeability of monolayer pulmonary vein VECs. This report shows that Cx43 participates in the regulation of vascular permeability in sepsis and that the mechanism is related to the Rock1-MLC20 phosphorylation pathway. PMID:26342084

  19. Attenuated Levels of Hippocampal Connexin 43 and its Phosphorylation Correlate with Antidepressant- and Anxiolytic-Like Activities in Mice

    PubMed Central

    Quesseveur, Gaël; Portal, Benjamin; Basile, Jean-Arnaud; Ezan, Pascal; Mathou, Alexia; Halley, Hélène; Leloup, Corinne; Fioramonti, Xavier; Déglon, Nicole; Giaume, Christian; Rampon, Claire; Guiard, Bruno P.

    2015-01-01

    Clinical and preclinical studies have implicated glial anomalies in major depression. Conversely, evidence suggests that the activity of antidepressant drugs is based, at least in part, on their ability to stimulate density and/or activity of astrocytes, a major glial cell population. Despite this recent evidence, little is known about the mechanism(s) by which astrocytes regulate emotionality. Glial cells communicate with each other through gap junction channels (GJCs), while they can also directly interact with neurons by releasing gliotransmitters in the extracellular compartment via an hemichannels (HCs)-dependent process. Both GJCs and HCs are formed by two main protein subunits: connexins (Cx) 30 and 43 (Cx30 and Cx43). Here we investigate the role of hippocampal Cx43 in the regulation of depression-like symptoms using genetic and pharmacological approaches. The first aim of this study was to evaluate the impact of the constitutive knock-down of Cx43 on a set of behaviors known to be affected in depression. Conversely, the expression of Cx43 was assessed in the hippocampus of mice subjected to prolonged corticosterone (CORT) exposure, given either alone or in combination with an antidepressant drug, the selective serotonin reuptake inhibitor fluoxetine. Our results indicate that the constitutive deficiency of Cx43 resulted in the expression of some characteristic hallmarks of antidepressant-/anxiolytic-like behavioral activities along with an improvement of cognitive performances. Moreover, in a new cohort of wild-type mice, we showed that CORT exposure elicited anxiety and depression-like abnormalities that were reversed by chronic administration of fluoxetine. Remarkably, CORT also increased hippocampal amounts of phosphorylated form of Cx43 whereas fluoxetine treatment normalized this parameter. From these results, we envision that antidepressant drugs may exert their therapeutic activity by decreasing the expression and/or activity of Cx43 resulting from a lower level of phosphorylation in the hippocampus. PMID:26733815

  20. Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo

    PubMed Central

    Basu, Rahul; Banerjee, Kaveri; Bose, Abhishek

    2015-01-01

    ABSTRACT Gap junctions (GJs) form intercellular channels which directly connect the cytoplasm between neighboring cells to facilitate the transfer of ions and small molecules. GJs play a major role in the pathogenesis of infection-associated inflammation. Mutations of gap junction proteins, connexins (Cxs), cause dysmyelination and leukoencephalopathy. In multiple sclerosis (MS) patients and its animal model experimental autoimmune encephalitis (EAE), Cx43 was shown to be modulated in the central nervous system (CNS). The mechanism behind Cx43 alteration and its role in MS remains unexplored. Mouse hepatitis virus (MHV) infection-induced demyelination is one of the best-studied experimental animal models for MS. Our studies demonstrated that MHV infection downregulated Cx43 expression at protein and mRNA levels in vitro in primary astrocytes obtained from neonatal mouse brains. After infection, a significant amount of Cx43 was retained in endoplasmic reticulum/endoplasmic reticulum Golgi intermediate complex (ER/ERGIC) and GJ plaque formation was impaired at the cell surface, as evidenced by a reduction of the Triton X-100 insoluble fraction of Cx43. Altered trafficking and impairment of GJ plaque formation may cause the loss of functional channel formation in MHV-infected primary astrocytes, as demonstrated by a reduced number of dye-coupled cells after a scrape-loading Lucifer yellow dye transfer assay. Upon MHV infection, a significant downregulation of Cx43 was observed in the virus-infected mouse brain. This study demonstrates that astrocytic Cx43 expression and function can be modulated due to virus stress and can be an appropriate model to understand the basis of cellular mechanisms involved in the alteration of gap junction intercellular communication (GJIC) in CNS neuroinflammation. IMPORTANCE We found that MHV infection leads to the downregulation of Cx43 in vivo in the CNS. In addition, results show that MHV infection impairs Cx43 expression in addition to gap junction communication in primary astrocytes. After infection, Cx43 did not traffic normally to the membrane to form gap junction plaques, and that could be the basis of reduced functional gap junction coupling between astrocytes. This is an important first step toward understanding how viruses affect Cx43 expression and trafficking at the cellular level. This may provide a basis for understanding how structural alterations of astrocytic gap junctions can disrupt gap junction communication between other CNS cells in altered CNS environments due to infection and inflammation. More specifically, alteration of Cx43 may be the basis of the destabilization of Cx47 in oligodendrocytes seen in and around inflammatory demyelinating plaques in MS patients. PMID:26676788

  1. Connexin-43 gap junctions are involved in multiconnexin-expressing stromal support of hemopoietic progenitors and stem cells.

    PubMed

    Cancelas, J A; Koevoet, W L; de Koning, A E; Mayen, A E; Rombouts, E J; Ploemacher, R E

    2000-07-15

    Gap junctions (GJs) provide for a unique system of intercellular communication (IC) allowing rapid transport of small molecules from cell to cell. GJs are formed by a large family of proteins named connexins (Cxs). Cx43 has been considered as the predominantly expressed Cx by hematopoietic-supporting stroma. To investigate the role of the Cx family in hemopoiesis, we analyzed the expression of 11 different Cx species in different stromal cell lines derived from murine bone marrow (BM) or fetal liver (FL). We found that up to 5 Cxs are expressed in FL stromal cells (Cx43, Cx45, Cx30.3, Cx31, and Cx31.1), whereas only Cx43, Cx45, and Cx31 were clearly detectable in BM stromal cells. In vivo, the Cx43-deficient 14.5- to 15-day FL cobblestone area-forming cells (CAFC)-week 1-4 and colony-forming unit contents were 26%-38% and 39%-47% lower than in their wild-type counterparts, respectively. The reintroduction of the Cx43 gene into Cx43-deficient FL stromal cells was able to restore their diminished IC to the level of the wild-type FL stromal cells. In addition, these Cx43-reintroduced stromal cells showed an increased support ability (3.7-fold) for CAFC-week 1 in normal mouse BM and 5-fold higher supportive ability for CAFC-week 4 in 5-fluorouracil-treated BM cells as compared with Cx43-deficient FL stromal cells. These findings suggest that stromal Cx43-mediated IC, although not responsible for all GJ-mediated IC of stromal cells, plays a role in the supportive ability for hemopoietic progenitors and stem cells. (Blood. 2000;96:498-505) PMID:10887111

  2. Attenuated Levels of Hippocampal Connexin 43 and its Phosphorylation Correlate with Antidepressant- and Anxiolytic-Like Activities in Mice.

    PubMed

    Quesseveur, Gaël; Portal, Benjamin; Basile, Jean-Arnaud; Ezan, Pascal; Mathou, Alexia; Halley, Hélène; Leloup, Corinne; Fioramonti, Xavier; Déglon, Nicole; Giaume, Christian; Rampon, Claire; Guiard, Bruno P

    2015-01-01

    Clinical and preclinical studies have implicated glial anomalies in major depression. Conversely, evidence suggests that the activity of antidepressant drugs is based, at least in part, on their ability to stimulate density and/or activity of astrocytes, a major glial cell population. Despite this recent evidence, little is known about the mechanism(s) by which astrocytes regulate emotionality. Glial cells communicate with each other through gap junction channels (GJCs), while they can also directly interact with neurons by releasing gliotransmitters in the extracellular compartment via an hemichannels (HCs)-dependent process. Both GJCs and HCs are formed by two main protein subunits: connexins (Cx) 30 and 43 (Cx30 and Cx43). Here we investigate the role of hippocampal Cx43 in the regulation of depression-like symptoms using genetic and pharmacological approaches. The first aim of this study was to evaluate the impact of the constitutive knock-down of Cx43 on a set of behaviors known to be affected in depression. Conversely, the expression of Cx43 was assessed in the hippocampus of mice subjected to prolonged corticosterone (CORT) exposure, given either alone or in combination with an antidepressant drug, the selective serotonin reuptake inhibitor fluoxetine. Our results indicate that the constitutive deficiency of Cx43 resulted in the expression of some characteristic hallmarks of antidepressant-/anxiolytic-like behavioral activities along with an improvement of cognitive performances. Moreover, in a new cohort of wild-type mice, we showed that CORT exposure elicited anxiety and depression-like abnormalities that were reversed by chronic administration of fluoxetine. Remarkably, CORT also increased hippocampal amounts of phosphorylated form of Cx43 whereas fluoxetine treatment normalized this parameter. From these results, we envision that antidepressant drugs may exert their therapeutic activity by decreasing the expression and/or activity of Cx43 resulting from a lower level of phosphorylation in the hippocampus. PMID:26733815

  3. A Functional Assay to Assess Connexin 43-Mediated Cell-to-Cell Communication of Second Messengers in Cultured Bone Cells.

    PubMed

    Stains, Joseph P; Civitelli, Roberto

    2016-01-01

    Cell-to-cell transfer of small molecules is a fundamental way by which multicellular organisms coordinate function. Recent work has highlighted the complexity of biologic responses downstream of gap junctions. As the connexin-regulated effectors are coming into focus, there is a need to develop functional assays that allow specific testing of biologically relevant second messengers. Here, we describe a modification of the classic gap junction parachute assay to assess biologically relevant molecules passed through gap junctions. PMID:27207296

  4. Connexin26 Mutations Causing Palmoplantar Keratoderma and Deafness Interact with Connexin43, Modifying Gap Junction and Hemichannel Properties.

    PubMed

    Shuja, Zunaira; Li, Leping; Gupta, Shashank; Meşe, Gülistan; White, Thomas W

    2016-01-01

    Mutations in GJB2 (connexin [Cx]26) cause either deafness or deafness associated with skin diseases. That different disorders can be caused by distinct mutations within the same gene suggests that unique channel activities are influenced by each class of mutation. We have examined the functional characteristics of two human mutations, Cx26-H73R and Cx26-S183F, causing palmoplantar keratoderma (PPK) and deafness. Both failed to form gap junction channels or hemichannels when expressed alone. Coexpression of the mutants with wild-type Cx43 showed a transdominant inhibition of Cx43 gap junction channels, without reductions in Cx43 protein synthesis. In addition, the presence of mutant Cx26 shifted Cx43 channel gating and kinetics toward a more Cx26-like behavior. Coimmunoprecipitation showed Cx43 being pulled down more efficiently with mutant Cx26 than wild-type, confirming the enhanced formation of heteromeric connexons. Finally, the formation of heteromeric connexons resulted in significantly increased Cx43 hemichannel activity in the presence of Cx26 mutants. These findings suggest a common mechanism whereby Cx26 mutations causing PPK and deafness transdominantly influence multiple functions of wild-type Cx43. They also implicate a role for aberrant hemichannel activity in the pathogenesis of PPK and further highlight an emerging role for Cx43 in genetic skin diseases.

  5. Connexin43 Hemichannels Mediate Small Molecule Exchange between Chondrocytes and Matrix in Biomechanically-Stimulated Temporomandibular Joint Cartilage

    PubMed Central

    Zhang, Jing; Zhang, Hongyun; Zhang, Mian; Qiu, Zhongying; Wu, Yaoping; Callaway, Danielle A.; Jiang, Jean X.; Lu, Lei; Jing, Lei; Yang, Ting; Wang, Meiqing

    2015-01-01

    Objective Connexin (Cx) 43 hemichannels play a role in mechanotransduction. This study was undertaken in order to determine if Cx43 hemichannels were activated in rat temporomandibular joint (TMJ) chondrocytes under mechanical stimulation. Methods Sprague-Dawley rats were stimulated dental-mechanically. Cx43 expression in rat TMJ cartilage was determined with immunohistochemistry and real-time PCR, and Cx43 hemichannel opening was evaluated by the extra- and intracellular levels of prostaglandin E2 (PGE2). Both primary rat chondrocytes and ATDC5 cells were treated with fluid flow shear stress (FFSS) to induce hemichannel opening. The Cx43 expression level was then determined by real-time PCR or western blotting, and the extent of Cx43 hemichannel opening was evaluated by measuring both PGE2 release and cellular dye uptake. Results Cx43 expression and intra- and extracellular PGE2 levels were increased in mechanically-stimulated rat TMJ cartilage compared to the unstimulated control. The FFSS treatment increased Cx43 expression and induced Cx43 hemichannel opening in primary rat chondrocytes and ATDC5 cells indicated by enhanced PGE2 release and dye uptake. Furthermore, the Cx43 hemichannel opening could be blocked by the addition of 18β-glycyrrhetinic acid, a connexin channel inhibitor, Cx43-targeting siRNA, or by withdrawal of FFSS stimulation. The migration of cytosolic Cx43 protein to the plasma membrane in ATDC5 cells was still significant after 8h post 2-h FFSS treatment, and the Cx43 protein level was still high at 48h which returned to control levels at 72h after treatment. Conclusion Cx43 hemichannels are activated and mediate small molecule exchange between TMJ chondrocytes and matrix under mechanical stimulation. PMID:24704497

  6. Development of poly(3-octylthiophene) thin films for regulating osteoblast growth

    NASA Astrophysics Data System (ADS)

    Rincon-Rosenbaum, Charlene

    The investigation of electrically conducting polymers (CPs) for use in biomedical applications has expanded greatly since the discovery in the 1980s that these materials are compatible with many biological molecules. CPs are able, via electrical stimulation, to modulate the behavior of certain electrically responsive cells (i.e., nerve, muscle, bone, and cardiac cells). CPs such as polypyrrole, polyaniline, and polythiophene have a conjugated structure that upon doping allows interchain hopping of electrons. In addition, most CPs have numerous attractive properties for biomedical applications, including the ability to transfer charges, to entrap and release biological molecules, and the potential to vary their chemical, electrical, and physical properties. Even though there has been significant progress, many biomedical issues remain unexplored, especially the interaction between different cell types (e.g., neurons, fibroblasts, and osteoblasts) and substituted polythiophenes (PTs) in both the undoped and doped states. PTs are one of the most widely studied CPs, therefore ample knowledge exists on their chemical, electrical, and physical properties. They also have great potential for biomedical applications as they have been used as biosensors, molecular actuators, and cell support substrates. The overall objective of this work is to assess the suitability of poly(3-octylthiophene) (P3OT) to sustain MC3T3-E1 osteoblast attachment and growth. The central hypothesis is that specific P3OT film properties (e.g., thickness, film preparation conditions, and level of doping) are able to regulate osteoblast functions (e.g., attachment and proliferation). Discrete and combinatorial techniques were utilized in this work to prepare and characterize thin films of P3OT, a semiconductor in its undoped state, and to study its interaction with MC3T3-E1 osteoblasts. The MC3T3-E1 cell line was chosen because it is well understood, is known to exhibit a developmental sequence

  7. Sulfated glycosaminoglycans support osteoblast functions and concurrently suppress osteoclasts.

    PubMed

    Salbach-Hirsch, Juliane; Ziegler, Nicole; Thiele, Sylvia; Moeller, Stephanie; Schnabelrauch, Matthias; Hintze, Vera; Scharnweber, Dieter; Rauner, Martina; Hofbauer, Lorenz C

    2014-06-01

    In order to improve bone regeneration, development and evaluation of new adaptive biomaterials is warranted. Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are major extracellular matrix (ECM) components of bone, and display osteogenic properties that are potentially useful for biomaterial applications. Using native and synthetic sulfate-modified GAGs, we manufactured artificial collagen/GAG ECM (aECMs) coatings, and evaluated how the presence of GAGs and their degree of sulfation affects the differentiation of murine mesenchymal stem cells to osteoblasts (OB) cultivated on these aECMs. GAG sulfation regulated osteogenesis at all key steps of OB development. Adhesion, but not migration, was diminished by 50% (P < 0.001). Proliferation and metabolic activity were slightly (P < 0.05) and cell death events strongly (P < 0.001) down-regulated due to a switch from proliferative to matrix synthesis state. When exposed to sulfated GAGs, OB marker genes, such as alkaline phosphatase, osteoprotegerin (OPG), and osteocalcin increased by up to 28-fold (P < 0.05) and calcium deposition up to 4-fold (P < 0.05). Furthermore, GAG treatment of OBs suppressed their ability to support osteoclast (OC) differentiation and resorption. In conclusion, GAG sulfation controls bone cell homeostasis by concurrently promoting osteogenesis and suppressing their paracrine support of OC functions, thus displaying a favorable profile on bone remodeling. Whether these cellular properties translate into improved bone regeneration needs to be validated in vivo.

  8. Pinctada fucata mantle gene 3 (PFMG3) promotes differentiation in mouse osteoblasts (MC3T3-E1).

    PubMed

    Wang, Xiaoyan; Liu, Shangfeng; Xie, Liping; Zhang, Rongqing; Wang, Zhao

    2011-02-01

    Nacre is secreted from the mantle of pearl oysters. In vivo and in vitro experiments have demonstrated that water-soluble extracts of nacre stimulate osteoblast differentiation and matrix mineralization, but the component responsible for this activity is unclear. It was reported that Pinctada fucata mantle gene 3 (PFMG3) with an N-terminal signal peptide could be secreted into the nacre of P. fucata. Here we report that PFMG3 is specifically expressed at the outer fold of the mantle and could promote calcium carbonate crystal formation in vitro. Consistent with this observation, we found that matrix mineralization of MC3T3-E1 cells, a murine osteoblast cell line, is accelerated upon treatment with PFMG3. Intriguingly, we observed that alkaline phosphatase activity and cell viability are increased after treating MC3T3-E1 cell with PFMG3. mRNA levels of osteoblast-specific marker genes osteocalcin and osteopontin are also increased. We conclude that PFMG3 from the mantle of P. fucata promotes MC3T3-E1 osteoblast cell differentiation, matrix mineralization, and calcium carbonate deposition in vitro. Our findings provide new evidence that PFMG3 may be used as a potential therapeutic molecule for the treatment of osteoporosis.

  9. Nano-hydroxyapatite modulates osteoblast lineage commitment by stimulation of DNA methylation and regulation of gene expression

    PubMed Central

    Ha, Shin-Woo; Jang, Hae Lin; Nam, Ki Tae; Beck, George R.

    2015-01-01

    Hydroxyapatite (HA) is the primary structural component of the skeleton and dentition. Under biological conditions, HA does not occur spontaneously and therefore must be actively synthesized by mineralizing cells such as osteoblasts. The mechanism(s) by which HA is actively synthesized by cells and deposited to create a mineralized matrix are not fully understood and the consequences of mineralization on cell function are even less well understood. HA can be chemically synthesized (HAp) and is therefore currently being investigated as a promising therapeutic biomaterial for use as a functional scaffold and implant coating for skeletal repair and dental applications. Here we investigated the biological effects of nano-HAp (10×100 nm) on the lineage commitment and differentiation of bone forming osteoblasts. Exposure of early stage differentiating osteoblasts resulted in dramatic and sustained changes in gene expression, both increased and decreased, whereas later stage osteoblasts were much less responsive. Analysis of the promoter region one of the most responsive genes, alkaline phosphatase, identified the stimulation of DNA methylation following cell exposure to nano-HAp. Collectively, the results reveal the novel epigenetic regulation of cell function by nano-HAp which has significant implication on lineage determination as well as identifying a novel potential therapeutic use of nanomaterials. PMID:26141836

  10. DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

    SciTech Connect

    Xu, Yong; Fang, Shi-ji; Zhu, Li-juan; Zhu, Lun-qing; Zhou, Xiao-zhong

    2014-10-24

    Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

  11. Interaction between Schwann Cells and Osteoblasts In Vitro

    PubMed Central

    Cai, Xiao-xiao; Luo, En; Yuan, Quan

    2010-01-01

    Aim Given the well-known properties of Schwann cells in promoting nerve regeneration, transplanting Schwann cells into implant sockets might be an effective method to promote sensory responses of osseointegrated implants. The aim of this study was to evaluate the interaction between Schwann cells and osteoblasts. Methodology Schwann cells derived from the sciatic nerves of neonatal rat were co-culured with osteoblasts using Transwell inserts. The proliferation of Schwann cells in the co-culture system was evaluated using methylthiazol tetrazolium (MTT) colorimetric method. Moreover, the secretions and mRNA levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR, respectively. In order to test the effect of Schwann cells on osteoblasts, alkaline phosphatase (ALP) staining and Alizerin red staining were performed as well. Results Schwann cells, which were co-cultured with the osteoblasts, showed an intact proliferation during the observation period. Moreover, the gene expression and synthesis of BDNF and NGF were not impaired by the osteoblasts. Meanwhile, co-cultured osteoblasts exhibited a significant increase in the proliferation on day 3 and 6 (P< 0.05). Co-culture of these two types of cells also led to a more intense staining of ALP and an elevated number of calcified nodules. Conclusion These findings demonstrate that, in the in vitro indirect co-culture environment, Schwann cells can maintain their normal ability to synthesize neurotrophins, which then enhance the proliferation and differentiation of osteoblasts. PMID:20737933

  12. Osteoblast hydraulic conductivity is regulated by calcitonin and parathyroid hormone

    NASA Technical Reports Server (NTRS)

    Hillsley, M. V.; Frangos, J. A.

    1996-01-01

    It is our hypothesis that osteoblasts play a major role in regulating bone (re)modeling by regulating interstitial fluid (ISF) flow through individual bone compartments. We hypothesize that osteoblasts of the blood-bone membrane lining the bone surfaces are capable of regulating transosseous fluid flow. This regulatory function of the osteoblasts was tested in vitro by culturing a layer of rat calvarial osteoblasts on porous membranes. Such a layer of osteoblasts subjected to 7.3 mm Hg of hydrostatic pressure posed a significant resistance to fluid flow across the cell layer similar in magnitude to the resistance posed by endothelial monolayers in vitro. The hydraulic conductivity, the volumetric fluid flux per unit pressure drop, of the osteoblast layer was altered in response to certain hormones. Hydraulic conductivity decreased approximately 40% in response to 33 nM parathyroid hormone, while it exhibited biphasic behavior in response to calcitonin: increased 40% in response to 100 nM calcitonin and decreased 40% in response to 1000 nM calcitonin. Further, activation of adenylate cyclase by forskolin dramatically increased the hydraulic conductivity, while elevation of intracellular calcium, [Ca2+]i, by the calcium ionophore A23187 initially decreased the hydraulic conductivity at 5 minutes before increasing conductivity by 30 minutes. These results suggest that cyclic adenosine monophosphate (cAMP) and [Ca2+]i may mediate changes in the osteoblast hydraulic conductivity. The increase in hydraulic conductivity in response to 100 nM calcitonin and the decrease in response to PTH suggest that the stimulatory and inhibitory effects on bone formation of calcitonin and parathyroid hormone, respectively, may be due in part to alterations in bone fluid flow.

  13. Osteoblasts detect pericellular calcium concentration increase via neomycin-sensitive voltage gated calcium channels.

    PubMed

    Sun, Xuanhao; Kishore, Vipuil; Fites, Kateri; Akkus, Ozan

    2012-11-01

    intracellular calcium occurs by the entry of extracellular calcium ions through VGCCs which are sensitive to neomycin. N-type and P-type VGCCs are potential candidates because they are observed in osteoblasts and they are sensitive to neomycin. The calcium channels identified in this study provide new insight into mechanisms underlying the targeted repair process which is essential to bone adaptation.

  14. Inhibitors of Growth 1b Suppresses Peroxisome Proliferator-Activated Receptor-β/δ Expression Through Downregulation of Hypoxia-Inducible Factor 1α in Osteoblast Differentiation.

    PubMed

    Qu, Bo; Hong, Zhen; Gong, Kai; Sheng, Jun; Wu, Hong-Hua; Deng, Shao-Lin; Huang, Gang; Ma, Ze-Hui; Pan, Xian-Ming

    2016-04-01

    Bone formation, a highly regulated developmental process, involves osteoblast differentiation, which is controlled by different important transcription factors. Recent evidence has suggested possible negative regulation of inhibitors of growth (ING) 1b on the osteoblast marker expression. The aim of this study is to examine the detailed mechanism by which the activity of ING1b inhibits osteoblast differentiation. In the current study, we investigated the function and mechanism by which ING1b inhibits osteoblast differentiation using C3H10T1/2 mesenchymal stem cells and MC3T3-E1 preosteoblasts. Real-time polymerase chain reaction and Western blotting showed that ING1b was decreased during osteoblast differentiation and ING1b overexpression markedly decreased alkaline phosphatase (ALP) activity, runt-related transcription factor 2 (Runx2) expression, and collagen type 1 synthesis, whereas ING1b silencing significantly upregulated ALP activity, Runx2 expression, and collagen type 1 synthesis. Further studies indicated that ING1b suppressed the expression of peroxisome proliferator-activated receptor (PPAR)-β/δ in a hypoxia-inducible factor (HIF) 1α-dependent manner, while ING1b silencing significantly increased the expression of PPAR-β/δ and HIF1α. Moreover, PPAR-β/δ or HIF1α silencing significantly inhibited ALP activity, Runx2 expression, and collagen type 1 synthesis. These results demonstrated that ING1b is an important regulator of osteoblast differentiation and suppresses PPAR-β/δ. Our study may provide additional insight into osteoblast differentiation and offer a potential new molecular target for osteoporosis. PMID:26849833

  15. MicroRNA-218, microRNA-191*, microRNA-3070a and microRNA-33 are responsive to mechanical strain exerted on osteoblastic cells.

    PubMed

    Guo, Yong; Wang, Yang; Liu, Yinqin; Liu, Yongming; Zeng, Qiangcheng; Zhao, Yumin; Zhang, Xinchang; Zhang, Xizheng

    2015-08-01

    MicroRNA (miRNA) is an important regulator of cell differentiation and function. Mechanical strain is important in the growth and differentiation of osteoblasts. Therefore, mechanresponsive miRNA may be important in the response of osteoblasts to mechanical strain. The purpose of the present study was to select and identify the mechanoresponsive miRNAs of osteoblasts. Mouse osteoblastic MC3T3-E1 cells were cultured in cell culture dishes and stimulated with a mechanical tensile strain of 2,50 με at 0.5 Hz, and the activity of alkaline phosphatase (ALP), mRNA levels of ALP, osteocalcin (OCN), and collagen type I (Col I), and protein levels of bone morphogenetic proteins (BMPs) in the cell culture medium were assayed. Following miRNA microarray and reverse transcription-quantitative polymerase chain reaction analyses, differentially expressed miRNAs in the mechanically strained cells and unstrained cells were selected and identified. Using bioinformatics analysis, the target genes of the miRNAs were then predicted. The results revealed that the mechanical strain of 2,500 με increased the activity of ALP, the mRNA levels of ALP, OCN and Col I, and the protein levels of bone morphogenetic protein(BMP)-2 and BMP-4 Continuous mechanical stimulation for 8 h had the most marked stimulant effects. miR-218, miR-191*, miR-3070a and miR-33 were identified as differentially expressed miRNAs in the mechanically strained MC3T3-E1 cells. Certain target genes of these four miRNAs were involved in osteoblastic differentiation. These findings indicated that a mechanical strain of 2,500 με, particularly for a period of 8 h, promoted osteoblastic differentiation, and the four mechanoresponsive miRNAs identified may be a potential regulator of osteoblastic differentiation and their response to mechanical strain.

  16. OSTEOBLAST ADHESION OF BREAST CANCER CELLS WITH SCANNING ACOUSTIC MICROSCOPY

    SciTech Connect

    Chiaki Miyasaka; Robyn R. Mercer; Andrea M. Mastro; Ken L. Telschow

    2005-03-01

    Breast cancer frequently metastasizes to the bone. Upon colonizing bone tissue, the cancer cells stimulate osteoclasts (cells that break bone down), resulting in large lesions in the bone. The breast cancer cells also affect osteoblasts (cells that build new bone). Conditioned medium was collected from a bone-metastatic breast cancer cell line, MDA-MB-231, and cultured with an immature osteoblast cell line, MC3T3-E1. Under these conditions the osteoblasts acquired a changed morphology and appeared to adherer in a different way to the substrate and to each other. To characterize cell adhesion, MC3T3-E1 osteoblasts were cultured with or without MDA-MB-231 conditioned medium for two days, and then assayed with a mechanical scanning acoustic reflection microscope (SAM). The SAM indicated that in normal medium the MC3T3-E1 osteoblasts were firmly attached to their plastic substrate. However, MC3T3-E1 cells cultured with MDA-MB-231 conditioned medium displayed both an abnormal shape and poor adhesion at the substrate interface. The cells were fixed and stained to visualize cytoskeletal components using optical microscopic techniques. We were not able to observe these differences until the cells were quite confluent after 7 days of culture. However, using the SAM, we were able to detect these changes within 2 days of culture with MDA-MB-231 conditioned medium

  17. Dynamics of the Transition from Osteoblast to Osteocyte

    PubMed Central

    Dallas, Sarah L.; Bonewald, Lynda F.

    2010-01-01

    Osteocytes are derived from osteoblasts and make up over ninety percent of the cells in bone. However, the mechanisms that control the differentiation of osteoblasts into osteocytes embedded in bone matrix are not well understood. With the recent development of transgenic models for manipulating gene expression in osteocytes and transgenic mice carrying lineage reporters for osteoblasts and osteocytes, unprecedented new insights are becoming possible. This article will review recent advances, such as comparative gene and protein expression studies that are delineating the changes in gene and protein expression that accompany osteocyte differentiation. It will also review recent studies in which time lapse dynamic imaging approaches have been used to visualize osteoblast and osteocyte populations within bone. These approaches reveal the key role of cell motility in bone cell function and highlight the dynamic nature of mineralized tissues. Changes in motile properties of the cell may be key in the transition from osteoblast to osteocyte, as reflected in the altered expression of many molecules involved in cytoskeletal function. PMID:20392270

  18. Repercussions of NSAIDS drugs on bone tissue: the osteoblast.

    PubMed

    García-Martínez, O; De Luna-Bertos, E; Ramos-Torrecillas, J; Manzano-Moreno, F J; Ruiz, C

    2015-02-15

    Non-steroidal anti-inflammatory drugs (NSAIDs) can act by modulating the behavior of osteoblasts, including their proliferation, differentiation, adhesion, and migration, but not all NSAIDs have these effects. Our objective was to update the information on this issue in a review of the literature in order to offer guidance on the prescription of the appropriate NSAID(s) to patients requiring bone tissue repair. To review current knowledge of this issue by searching for all relevant publications since 2001 in the MEDLINE, EMBASE and Cochrane Library databases, we used the following descriptors: bone tissue, osteoblast, NSAIDs, Anti-inflammatory drugs. Published studies show that most NSAIDs have an adverse effect on osteoblast growth by cell cycle arrest and apoptosis induction. The effect on differentiation varies according to the drug, dose, and treatment time. Osteoblast adhesion is increased and migration decreased by some NSAIDs, such as indomethacin and diclofenac. The antigenic profile or phagocytic function can also be modulated by NSAIDs. In general, NSAIDs have an adverse effect on bone tissue and given the routine administration of NSAIDs to individuals requiring bone repair, in which the osteoblast has an essential role, this effect on bone should be borne in mind.

  19. Knockdown of PRKAR1A, the gene responsible for Carney complex, interferes with differentiation in osteoblastic cells.

    PubMed

    Zhang, Mei; Manchanda, Parmeet K; Wu, Dayong; Wang, Qianben; Kirschner, Lawrence S

    2014-03-01

    PRKAR1A is the gene encoding the type 1A regulatory subunit of protein kinase A, and it is the cause of the inherited human tumor syndrome Carney complex. Data from our laboratory has demonstrated that Prkar1a loss causes tumors in multiple cell lineages, including neural crest cells and osteoblasts. We have proposed that one mechanism by which tumorigenesis occurs is through the failure of terminal differentiation. In the present study, we directly test the effects of Prkar1a reduction on osteogenic differentiation in mouse and human cells in vitro. We found that Prkar1a levels noticeably increased during osteoblastic differentiation, indicating a positive correlation between the expression of Prkar1a and osteogenic potential. To validate this hypothesis, we generated stable Prkar1a knockdown in both mouse and human cells. These cells displayed significantly suppressed bone nodule formation and decreased expression of osteoblast markers such as osteocalcin and osteopontin. These observations imply that the antiosteogenic effect of Prkar1a ablation is not species or cell line specific. Furthermore, because Runt-related transcription factor-2 (Runx2) is a key mediator of osteoblast differentiation, we reasoned that the function of this transcription factor may be inhibited by Prkar1a knockdown. Chromatin immunoprecipitation and luciferase assays demonstrated that Prkar1a ablation repressed DNA binding and function of Runx2 at its target genes. Additionally, we determined that this effect is likely due to reductions in the Runx2-cooperating transcription factors forkhead box O1 and activating transcription factor 4. Taken together, this study provides direct evidence that ablation of Prkar1a interferes with signaling pathways necessary for osteoblast differentiation. PMID:24506536

  20. Biphasic Response to Luteolin in MG-63 Osteoblast-Like Cells under High Glucose-Induced Oxidative Stress

    PubMed Central

    Abbasi, Naser; Khosravi, Afra; Aidy, Ali; Shafiei, Massoumeh

    2016-01-01

    Background: Clinical evidence indicates the diabetes-induced impairment of osteogenesis caused by a decrease in osteoblast activity. Flavonoids can increase the differentiation and mineralization of osteoblasts in a high-glucose state. However, some flavonoids such as luteolin may have the potential to induce cytotoxicity in osteoblast-like cells. This study was performed to investigate whether a cytoprotective concentration range of luteolin could be separated from a cytotoxic concentration range in human MG-63 osteoblast-like cells in high-glucose condition. Methods: Cells were cultured in a normal- or high-glucose medium. Cell viability was determined with the MTT assay. The formation of intracellular reactive oxygen species (ROS) was measured using probe 2’,7’ -dichlorofluorescein diacetate, and osteogenic differentiation was evaluated with an alkaline phosphatase bioassay. Results: ROS generation, reduction in alkaline phosphatase activity, and cell death induced by high glucose were inhibited by lower concentrations of luteolin (EC50, 1.29±0.23 µM). Oxidative stress mediated by high glucose was also overcome by N-acetyl-L-cysteine. At high concentrations, luteolin caused osteoblast cell death in normal- and high-glucose states (IC50, 34±2.33 and 27±2.42 µM, respectively), as represented by increased ROS and decreased alkaline phosphatase activity. Conclusion: Our results indicated that the cytoprotective action of luteolin in glucotoxic condition was manifested in much lower concentrations, by a factor of approximately 26 and 20, than was its cytotoxic activity, which occurred under normal or glucotoxic condition, respectively. PMID:26989282

  1. Correlative spectroscopy of silicates in mineralised nodules formed from osteoblasts

    NASA Astrophysics Data System (ADS)

    Boonrungsiman, Suwimon; Fearn, Sarah; Gentleman, Eileen; Spillane, Liam; Carzaniga, Raffaella; McComb, David W.; Stevens, Molly M.; Porter, Alexandra E.

    2013-07-01

    Silicon supplementation has been shown to play an important role in skeleton development, however, the potential role that silicon plays in mediating bone formation, and an understanding of where it might localise in the resulting bone tissue remain elusive. An improved understanding of these processes could have important implications for treating pathological mineralisation. A key aspect of defining the role of silicon in bone is to characterise its distribution and coordination environment, however, there is currently almost no information available on either. We have combined a sample-preparation method that simultaneously preserved mineral, ions, and the extracellular matrix (ECM) with secondary ion mass spectroscopy (SIMS) and electron energy-loss spectroscopy (EELS) to examine the distribution and coordination environment of silicon in murine osteoblasts (OBs) in an in vitro model of bone formation. SIMS analysis showed a high level of surface contamination from polydimethysiloxane (PDMS) resulting from sample preparation. When the PDMS was removed, silicon compounds could not be detected within the nodules either by SIMS or by energy dispersive X-ray spectroscopy (EDX) analysis. In comparison, electron energy-loss spectroscopy (EELS) provided a powerful and potentially widely applicable means to define the coordination environment and localisation of silicon in mineralising tissues. We show that trace levels of silicon were only detectable from the mineral deposits located on the collagen and in the peripheral region of mineralised matrix, possibly the newly mineralised regions of the OB nodules. Taken together our results suggest that silicon plays a biological role in bone formation, however, the precise mechanism by which silicon exerts its physicochemical effects remains uncertain. Our analytical results open the door for compelling new sets of EELS experiments that can provide detailed and specific information about the role that silicates play in bone

  2. Calcium signals and calcium channels in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Duncan, R. L.; Akanbi, K. A.; Farach-Carson, M. C.

    1998-01-01

    Calcium (Ca2+) channels are present in non-excitable as well as in excitable cells. In bone cells of the osteoblast lineage, Ca2+ channels play fundamental roles in cellular responses to external stimuli including both mechanical forces and hormonal signals. They are also proposed to modulate paracrine signaling between bone-forming osteoblasts and bone-resorbing osteoclasts at local sites of bone remodeling. Calcium signals are characterized by transient increases in intracellular Ca2+ levels that are associated with activation of intracellular signaling pathways that control cell behavior and phenotype, including patterns of gene expression. Development of Ca2+ signals is a tightly regulated cellular process that involves the concerted actions of plasma membrane and intracellular Ca2+ channels, along with Ca2+ pumps and exchangers. This review summarizes the current state of knowledge concerning the structure, function, and role of Ca2+ channels and Ca2+ signals in bone cells, focusing on the osteoblast.

  3. Osteoblastic differentiation of monkey embryonic stem cells in vitro.

    PubMed

    Yamashita, Akihiro; Takada, Tatsuyuki; Narita, Junko; Yamamoto, Gaku; Torii, Ryuzo

    2005-01-01

    Monkey embryonic stem (ES) cell is a useful tool for preclinical studies of regenerative medicine. In this paper, we investigated whether monkey ES cells can be differentiated into osteoblasts in vitro using factors known to promote osteogenesis. We prepared embryoid bodies (EB) in the presence of retinoic acid (RA) and subsequently differentiated in the medium containing either dexamethasone (DEX) or bone morphogenetic protein (BMP)-2 in addition to osteogenic supplements (OS), specifically ascorbic acid and beta-glycerophosphate. RA treatment during EB formation induced osteoblastic marker genes, such as collagen type 1, osteopontin, and Cbfa1. For the expression of osteocalcin, however, cultivation with medium containing either DEX or BMP-2 in addition to OS was required. These results showed that osteoblasts could be derived from monkey ES cells in vitro and BMP-2 + OS was effective to induce calcification. PMID:16390259

  4. Curcumin inhibits the proliferation and mineralization of cultured osteoblasts.

    PubMed

    Notoya, Michitaka; Nishimura, Hiroyuki; Woo, Je-Tae; Nagai, Kazuo; Ishihara, Yoko; Hagiwara, Hiromi

    2006-03-18

    The effects of curcumin, which is an important constituent of rhizomes of the plant Curcuma longa Linn, on the metabolism of osteoblasts were examined in cultures of rat calvarial osteoblastic cells (ROB cells). The proliferation of cells was markedly inhibited upon exposure of cells to curcumin at 5x10(-6) to 1x10(-5) M. Curcumin at 1x10(-5) M did not induce apoptosis in ROB cells but arrested cells at the G1 phase of the cell cycle. In addition, curcumin stimulated the expression of mRNA for p21(WAF1/CIP1), which inhibits the activity of cyclin-dependent kinases, and inhibited the phosphorylation of histone H1. Furthermore, curcumin reduced the rate of deposition of calcium and the formation of mineralized nodules. Our results indicate that curcumin might inhibit the proliferation and mineralization of osteoblastic cells through the expression of p21(WAF1/CIP1). PMID:16476424

  5. Role of syndecan-2 in osteoblast biology and pathology

    PubMed Central

    Mansouri, Rafik; Haÿ, Eric; Marie, Pierre J; Modrowski, Dominique

    2015-01-01

    Syndecans 1–4 are a family of transmembrane proteins composed of a core protein and glycosaminoglycan chains. Although the four syndecans have common functions, they appear to be connected to different signaling pathways, and their expression occurs in a cell- and development-specific pattern. In contrast to other syndecans, syndecan-2 expression increases during osteoblast differentiation. Mechanistically, syndecan-2 exerts multiple functions in cells of the osteoblast lineage as it serves as a co-receptor for fibroblast growth factors and Wnt proteins and controls cell adhesion, proliferation, differentiation and apoptosis. Recent studies indicate that syndecan-2 also contributes to osteosarcoma cell response to cytotoxic agents through interactions with Wnt/β-catenin signaling. Here we summarize our current understanding of the role of syndecan-2 in the control of osteoblast biology and pathology and discuss how syndecan-2 acts as a modulator of the bone cell microenvironment. PMID:25848534

  6. Cellular Factor XIIIA Transglutaminase Localizes in Caveolae and Regulates Caveolin-1 Phosphorylation, Homo-oligomerization and c-Src Signaling in Osteoblasts.

    PubMed

    Wang, Shuai; Kaartinen, Mari T

    2015-11-01

    Transglutaminases (TGs) are a family of widely distributed enzymes that catalyze protein crosslinking by forming a covalent isopeptide bond between the substrate proteins. We have shown that MC3T3-E1 osteoblasts express Factor XIII-A (FXIII-A), and that the extracellular crosslinking activity of FXIII-A is involved in regulating matrix secretion and deposition. In this study, we have investigated the localization and potential role of intracellular FXIII-A. Conventional immunofluorescence microscopy and TIRF microscopy analyses showed that FXIII-A co-localizes with caveolin-1 in specialized membrane structures, caveolae, in differentiating osteoblasts. The caveolae-disrupting agent methyl-β-cyclodextrin abolished FXIII-A staining and co-localization with caveolin-1 from the osteoblast plasma membrane. The presence of FXIII-A in caveolae was confirmed by preparing caveolae-enriched cellular fractions using sucrose density gradient ultracentrifugation followed by western blotting. Despite this association of FXIII-A with caveolae, there was no detectable transglutaminase activity in caveolae, as measured by monodansylcadaverine incorporation. TG inhibitor NC9--which can alter TG enzyme conformation--localized to caveolae and displaced FXIII-A from these structures when added to the osteoblast cultures. The decreased FXIII-A levels in caveolae after NC9 treatment increased c-Src activation, which resulted in caveolin-1 phosphorylation, homo-oligomerization and Akt phosphorylation, suggesting cellular FXIII-A has a role in regulating c-Src signaling in osteoblasts. PMID:26231113

  7. Enhanced osteoblast proliferation and collagen gene expression by estradiol

    SciTech Connect

    Ernest, M.; Schmid, Ch.; Froesch, E.R. )

    1988-04-01

    Estrogens play a crucial role in the development of postmenopausal osteoporosis. However, the mechanism by which estrogens exert their effects on bone is unknown. To examine possible direct effects of 17{beta}-estradiol on bone-forming cells, the authors used pure rat osteoblast-like cells in vitro as a model. Osteoblast-like cells prepared from calvaria of newborn rats were cultured serum-free in methylcellulose-containing medium for 21 days. Osteoblast-like cells proliferate selectively into clonally derived cell clusters of spherical morphorlogy. 17{beta}-Estradiol at concentrations of 0.1 nM and 1 nM enhanced osteoblast-like cell proliferation by 41% and 68% above vehicle-treated controls. The biologically inactive stereoisomer 17{alpha}-estradiol (same concentrations) had no effect. Moreover, the antiestrogen tamoxifen abolished the stimulation of osteoblast-like cell proliferation by 17{beta}-estradiol. After 21 days of culture, RNA was prepared and analyzed in a dot-hybridization assay for the abundance of pro{alpha}1(I) collagen mRNA. Steady-state mRNA levels were increased in cultures treated with 17{beta}-estradiol in a dose-dependent manner with maximal stimulation at 1 nM and 10 nM. At the same concentrations, the percentage of synthesized protein (labeled by ({sup 3}H)proline pulse) that was digestible by collagenase was increased, indicating that 17{beta}-estradiol acts as pretranslational levels to enhance synthesis of bone collagen. These data show that the osteoblast is a direct target for 17{beta}-estradiol.

  8. Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

    SciTech Connect

    Yonezawa, Takayuki; Lee, Ji-Won; Hibino, Ayaka; Asai, Midori; Hojo, Hironori; Cha, Byung-Yoon; Teruya, Toshiaki; Nagai, Kazuo; Chung, Ung-Il; Yagasaki, Kazumi; and others

    2011-06-03

    Highlights: {yields} Harmine promotes the activity and mRNA expression of ALP. {yields} Harmine enhances the expressions of osteocalcin mRNA and protein. {yields} Harmine induces osteoblastic mineralization. {yields} Harmine upregulates the mRNA expressions of BMPs, Runx2 and Osterix. {yields} BMP signaling pathways are involved in the actions of harmine. -- Abstract: Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a {beta}-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related {beta}-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of

  9. Changes in osteoblastic activity due to simulated weightless conditions

    NASA Technical Reports Server (NTRS)

    Doty, S. B.; Morey-Holton, E. R.

    1982-01-01

    Using histochemistry and electron microscopy, the reduced bone formation which occurs in the hypokinetic, orthostatically treated adult rat has been studied. The two major changes noted occurred in the osteoblast population, indicated by a reduced alkaline phosphatase activity and reduced numbers of gap junctions between cells. These results were most noticeable in the periosteum and endosteum of the long bones. Changes in osteoblasts lining the surface of trabecular bone were not as evident. These results indicate that the cells lining the surfaces of weight bearing bones are most affected by hypokinesia and this reduction in cellular activity may be a mechanically induced effect.

  10. Bioactive silica-based nanoparticles stimulate bone-forming osteoblasts, suppress bone-resorbing osteoclasts, and enhance bone mineral density in vivo.

    PubMed

    Beck, George R; Ha, Shin-Woo; Camalier, Corinne E; Yamaguchi, Masayoshi; Li, Yan; Lee, Jin-Kyu; Weitzmann, M Neale

    2012-08-01

    Bone is a dynamic tissue that undergoes renewal throughout life in a process whereby osteoclasts resorb worn bone and osteoblasts synthesize new bone. Imbalances in bone turnover lead to bone loss and development of osteoporosis and ultimately fracture, a debilitating condition with high morbidity and mortality. Silica is a ubiquitous biocontaminant that is considered to have high biocompatibility. The authors report that silica nanoparticles (NPs) mediate potent inhibitory effects on osteoclasts and stimulatory effects on osteoblasts in vitro. The mechanism of bioactivity is a consequence of an intrinsic capacity to antagonize activation of NF-κB, a signal transduction pathway required for osteoclastic bone resorption but inhibitory to osteoblastic bone formation. We further demonstrate that silica NPs promote a significant enhancement of bone mineral density (BMD) in mice in vivo, providing a proof of principle for the potential application of silica NPs as a pharmacological agent to enhance BMD and protect against bone fracture.

  11. Characterization of self-assembled decyl bis phosphonate-Collagen layers on titanium by QCM-D and osteoblast-compatibility

    NASA Astrophysics Data System (ADS)

    Ni, Yuxiang; Liu, Zhiyuan; Gao, Wenli; Qu, Shuxin; Weng, Jie; Feng, Bo

    2011-09-01

    Quartz crystal microbalance dissipation (QCM-D) was used to monitor the layer by layer (LBL) self-assemble process of decyl bis phosphonate (DBP) and Collagen on titanium. The mass and viscoelastic properties of self-assembled layers were obtained using QCM-D. The stability of DBP and Collagen layer on titanium was tested to be very good. Osteoblasts cell culture was performed on LBL modified samples and that after BSA adsorption. The morphology of cells was observed by a scanning electron microscope (SEM). The total metabolic activity and differentiation of osteoblasts were evaluated by a metabolic assay and alkaline phosphatase (ALP) activity, respectively. These tests showed that osteoblasts have better activity, proliferation, and differentiation on Collagen terminated samples and BSA adsorbed samples. These results, along with the good biomineralization and protein adsorption abilities of Ti/DBP/Collagen surface (tested in our previous work), suggest titanium modified by this layer by layer technique has the potential application for medical implants.

  12. Bisphosphonates modulate vital functions of human osteoblasts and affect their interactions with breast cancer cells.

    PubMed

    Kaiser, Tatjana; Teufel, Ingrid; Geiger, Konstanze; Vater, Yvonne; Aicher, Wilhelm K; Klein, Gerd; Fehm, Tanja

    2013-07-01

    Bisphosphonates (BPs) are in clinical use for the treatment of breast cancer patients with bone metastases. Their anti-resorptive effect is mainly explained by inhibition of osteoclast activity, but recent evidence also points to a direct action of BPs on bone-forming osteoblasts. However, the mechanisms how BPs influence osteoblasts and their interactions with breast cancer cells are still poorly characterized. Human osteoblasts isolated from bone specimens were characterized in depth by their expression of osteogenic marker genes. The influence of the nitrogen-containing BPs zoledronate (Zol), ibandronate (Iban), and pamidronate (Pam) on molecular and cellular functions of osteoblasts was assessed focusing on cell proliferation and viability, apoptosis, cytokine secretion, and osteogenic-associated genes. Furthermore, effects of BPs on osteoblast-breast tumor cell interactions were examined in an established in vitro model system. The BPs Zol and Pam inhibited cell viability of osteoblasts. This effect was mediated by an induction of caspase-dependent apoptosis in osteoblasts. By interfering with the mevalonate pathway, Zol also reduces the proliferation of osteoblasts. The expression of phenotypic markers of osteogenic differentiation was altered by Zol and Pam. In addition, both BPs strongly influenced the secretion of the chemokine CCL2 by osteoblasts. Breast cancer cells also responded to Zol and Pam with a reduced cell adhesion to osteoblast-derived extracellular matrix molecules and with a decreased migration in response to osteoblast-secreted factors. BPs revealed prominent effects on human osteoblasts. Zol and Pam as the most potent BPs affected not only the expression of osteogenic markers, osteoblast viability, and proliferation but also important osteoblast-tumor cell interactions. Changing the osteoblast metabolism by BPs modulates migration and adhesion of breast cancer cells as well. PMID:23807419

  13. Small molecule–driven direct conversion of human pluripotent stem cells into functional osteoblasts

    PubMed Central

    Kang, Heemin; Shih, Yu-Ru V.; Nakasaki, Manando; Kabra, Harsha; Varghese, Shyni

    2016-01-01

    The abilities of human pluripotent stem cells (hPSCs) to proliferate without phenotypic alteration and to differentiate into tissue-specific progeny make them a promising cell source for regenerative medicine and development of physiologically relevant in vitro platforms. Despite this potential, efficient conversion of hPSCs into tissue-specific cells still remains a challenge. Herein, we report direct conversion of hPSCs into functional osteoblasts through the use of adenosine, a naturally occurring nucleoside in the human body. The hPSCs treated with adenosine not only expressed the molecular signatures of osteoblasts but also produced calcified bone matrix. Our findings show that the adenosine-mediated osteogenesis of hPSCs involved the adenosine A2bR. When implanted in vivo, using macroporous synthetic matrices, the human induced pluripotent stem cell (hiPSC)–derived donor cells participated in the repair of critical-sized bone defects through the formation of neobone tissue without teratoma formation. The newly formed bone tissues exhibited various attributes of the native tissue, including vascularization and bone resorption. To our knowledge, this is the first demonstration of adenosine-induced differentiation of hPSCs into functional osteoblasts and their subsequent use to regenerate bone tissues in vivo. This approach that uses a physiologically relevant single small molecule to generate hPSC-derived progenitor cells is highly appealing because of its simplicity, cost-effectiveness, scalability, and impact in cell manufacturing, all of which are decisive factors for successful translational applications of hPSCs. PMID:27602403

  14. Small molecule-driven direct conversion of human pluripotent stem cells into functional osteoblasts.

    PubMed

    Kang, Heemin; Shih, Yu-Ru V; Nakasaki, Manando; Kabra, Harsha; Varghese, Shyni

    2016-08-01

    The abilities of human pluripotent stem cells (hPSCs) to proliferate without phenotypic alteration and to differentiate into tissue-specific progeny make them a promising cell source for regenerative medicine and development of physiologically relevant in vitro platforms. Despite this potential, efficient conversion of hPSCs into tissue-specific cells still remains a challenge. Herein, we report direct conversion of hPSCs into functional osteoblasts through the use of adenosine, a naturally occurring nucleoside in the human body. The hPSCs treated with adenosine not only expressed the molecular signatures of osteoblasts but also produced calcified bone matrix. Our findings show that the adenosine-mediated osteogenesis of hPSCs involved the adenosine A2bR. When implanted in vivo, using macroporous synthetic matrices, the human induced pluripotent stem cell (hiPSC)-derived donor cells participated in the repair of critical-sized bone defects through the formation of neobone tissue without teratoma formation. The newly formed bone tissues exhibited various attributes of the native tissue, including vascularization and bone resorption. To our knowledge, this is the first demonstration of adenosine-induced differentiation of hPSCs into functional osteoblasts and their subsequent use to regenerate bone tissues in vivo. This approach that uses a physiologically relevant single small molecule to generate hPSC-derived progenitor cells is highly appealing because of its simplicity, cost-effectiveness, scalability, and impact in cell manufacturing, all of which are decisive factors for successful translational applications of hPSCs. PMID:27602403

  15. Small molecule–driven direct conversion of human pluripotent stem cells into functional osteoblasts

    PubMed Central

    Kang, Heemin; Shih, Yu-Ru V.; Nakasaki, Manando; Kabra, Harsha; Varghese, Shyni

    2016-01-01

    The abilities of human pluripotent stem cells (hPSCs) to proliferate without phenotypic alteration and to differentiate into tissue-specific progeny make them a promising cell source for regenerative medicine and development of physiologically relevant in vitro platforms. Despite this potential, efficient conversion of hPSCs into tissue-specific cells still remains a challenge. Herein, we report direct conversion of hPSCs into functional osteoblasts through the use of adenosine, a naturally occurring nucleoside in the human body. The hPSCs treated with adenosine not only expressed the molecular signatures of osteoblasts but also produced calcified bone matrix. Our findings show that the adenosine-mediated osteogenesis of hPSCs involved the adenosine A2bR. When implanted in vivo, using macroporous synthetic matrices, the human induced pluripotent stem cell (hiPSC)–derived donor cells participated in the repair of critical-sized bone defects through the formation of neobone tissue without teratoma formation. The newly formed bone tissues exhibited various attributes of the native tissue, including vascularization and bone resorption. To our knowledge, this is the first demonstration of adenosine-induced differentiation of hPSCs into functional osteoblasts and their subsequent use to regenerate bone tissues in vivo. This approach that uses a physiologically relevant single small molecule to generate hPSC-derived progenitor cells is highly appealing because of its simplicity, cost-effectiveness, scalability, and impact in cell manufacturing, all of which are decisive factors for successful translational applications of hPSCs.

  16. NELL-1 increases pre-osteoblast mineralization using both phosphate transporter Pit1 and Pit2

    SciTech Connect

    Cowan, Catherine M.; Zhang, Xinli; James, Aaron W.; Mari Kim, T.; Sun, Nichole; Wu, Benjamin; Ting, Kang; Soo, Chia

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer NELL-1 accelerates extracellular matrix mineralization in MC3T3-E1 pre-osteoblasts. Black-Right-Pointing-Pointer NELL-1 significantly increases intracellular inorganic phosphate levels. Black-Right-Pointing-Pointer NELL-1 positively regulates osteogenesis but not proliferation in MC3T3-E1 cells. Black-Right-Pointing-Pointer NELL-1 regulates inorganic phosphate transporter activity. -- Abstract: NELL-1 is a potent osteoinductive molecule that enhances bone formation in multiple animal models through currently unidentified pathways. In the present manuscript, we hypothesized that NELL-1 may regulate osteogenic differentiation accompanied by alteration of inorganic phosphate (Pi) entry into the osteoblast via sodium dependent phosphate (NaPi) transporters. To determine this, MC3T3-E1 pre-osteoblasts were cultured in the presence of recombinant human (rh)NELL-1 or rhBMP-2. Analysis was performed for intracellular Pi levels through malachite green staining, Pit-1 and Pit-2 expression, and forced upregulation of Pit-1 and Pit-2. Results showed rhNELL-1 to increase MC3T3-E1 matrix mineralization and Pi influx associated with activation of both Pit-1 and Pit-2 channels, with significantly increased Pit-2 production. In contrast, Pi transport elicited by rhBMP-2 showed to be associated with increased Pit-1 production only. Next, neutralizing antibodies against Pit-1 and Pit-2 completely abrogated the Pi influx effect of rhNELL-1, suggesting rhNELL-1 is dependent on both transporters. These results identify one potential mechanism of action for rhNELL-1 induced osteogenesis and highlight a fundamental difference between NELL-1 and BMP-2 signaling.

  17. Mineralization and osteoblast response to bioactive glass in vitro.

    PubMed

    Zhou, Z H; Yi, Q F; Nei, H D; Ling, Y L; Zhou, J N; Liu, L H; Liu, X P

    2010-05-01

    Bioactive glass, an osteoproductive material, has received considerable attention as a bone graft substitute in the treatment of bony defects. Bioactive CaO-SiO(2)-P(2)O(5) glass was prepared using the sol-gel method, and mineralization behaviour in vitro was investigated by soaking it in simulated body fluid (SBF). Cellular cultivation in vitro, MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) and Von Kossa assays were conducted to evaluate the osteoblast response to the bioactive glass. A calcium phosphate carbonate hydroxide (HCA) layer was formed on the bioactive glass after soaking for 3 days in SBF, which indicated that the mineralization on the surface of bioactive glass could progress spontaneously. The osteoblast response results demonstrated that bioactive glass had no cytotoxicity, and it might not be harmful to the morphology of the osteoblast. The growth and proliferation of the osteoblastic cell could not be inhibited. Nodule formation was also observed in conditioned medium containing dissolution bioactive glass and these nodules were shown to be mineralized by Von Kossa staining, which indicates that bioactive glass shows good biocompatibility. PMID:20397850

  18. Allopurinol and oxypurinol promote osteoblast differentiation and increase bone formation

    PubMed Central

    Orriss, Isabel R.; Arnett, Timothy R.; George, Jacob; Witham, Miles D.

    2016-01-01

    Allopurinol and its active metabolite, oxypurinol are widely used in the treatment of gout and hyperuricemia. They inhibit xanthine oxidase (XO) an enzyme in the purine degradation pathway that converts xanthine to uric acid. This investigation examined the effect of allopurinol and oxypurinol on bone formation, cell number and viability, gene expression and enzyme activity in differentiating and mature, bone-forming osteoblasts. Although mRNA expression remained relatively constant, XO activity decreased over time with mature osteoblasts displaying reduced levels of uric acid (20% decrease). Treatment with allopurinol and oxypurinol (0.1–1 µM) reduced XO activity by up to 30%. At these concentrations, allopurinol and oxypurinol increased bone formation by osteoblasts ~4-fold and ~3-fold, respectively. Cell number and viability were unaffected. Both drugs increased tissue non-specific alkaline phosphatase (TNAP) activity up to 65%. Osteocalcin and TNAP mRNA expression was increased, 5-fold and 2-fold, respectively. Expression of NPP1, the enzyme responsible for generating the mineralisation inhibitor, pyrophosphate, was decreased 5-fold. Col1α1 mRNA expression and soluble collagen levels were unchanged. Osteoclast formation and resorptive activity were not affected by treatment with allopurinol or oxypurinol. Our data suggest that inhibition of XO activity promotes osteoblast differentiation, leading to increased bone formation in vitro. PMID:26968635

  19. Osteoblasts subjected to spaceflight and simulated space shuttle launch conditions.

    PubMed

    Kacena, Melissa A; Todd, Paul; Landis, William J

    2003-01-01

    To understand further the effects of spaceflight on osteoblast-enriched cultures, normal chicken calvarial osteoblasts were flown aboard shuttle flight STS-77, and the total number of attached cells was determined. Spaceflight and control cultures were chemically fixed 3 h and 3 d after launch. These fixed cultures were processed for scanning electron microscopy (SEM). The SEM analysis showed that with just 3 d of exposure to spaceflight, coverslip cultures contained 300 +/- 100 cells/mm2, whereas 1G control samples contained a confluent monolayer of cells (2400 +/- 200 cells/mm2). Although the cultures flown in space experienced a drastic decline in cell number in just 3 d, without further experimentation it was impossible to determine whether the decline was a result of microgravity, the harsh launch environment, or some combination of these factors. Therefore, this research attempted to address the effect of launch by subjecting osteoblasts to conditions simulating shuttle launch accelerations, noise, and vibrations. No differences, compared with controls, were seen in the number of total or viable cells after exposure to these various launch conditions. Taken together, these data indicate that the magnitude of gravitational loading (3G maximum) and vibration (7.83G rms maximum) resulting from launch does not adversely affect osteoblasts in terms of total or viable cell number immediately, but launch conditions, or the microgravity environment itself, may start a cascade of events that over several d contributes to cell loss.

  20. Genomic approaches to identifying transcriptional regulators of osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Stains, Joseph P.; Civitelli, Roberto

    2003-01-01

    Recent microarray studies of mouse and human osteoblast differentiation in vitro have identified novel transcription factors that may be important in the establishment and maintenance of differentiation. These findings help unravel the pattern of gene-expression changes that underly the complex process of bone formation.

  1. Wntless functions in mature osteoblasts to regulate bone mass.

    PubMed

    Zhong, Zhendong; Zylstra-Diegel, Cassandra R; Schumacher, Cassie A; Baker, Jacob J; Carpenter, April C; Rao, Sujata; Yao, Wei; Guan, Min; Helms, Jill A; Lane, Nancy E; Lang, Richard A; Williams, Bart O

    2012-08-14

    Recent genome-wide association studies of individuals of Asian and European descent have found that SNPs located within the genomic region (1p31.3) encoding the Wntless (Wls)/Gpr177 protein are associated significantly with reduced bone mineral density. Wls/Gpr177 is a newly identified chaperone protein that specifically escorts Wnt ligands for secretion. Given the strong functional association between the Wnt signaling pathways and bone development and homeostasis, we generated osteoblast-specific Wls-deficient (Ocn-Cre;Wls-flox) mice. Homozygous conditional knockout animals were born at a normal Mendelian frequency. Whole-body dual-energy X-ray absorptiometry scanning revealed that bone-mass accrual was significantly inhibited in homozygotes as early as 20 d of age. These homozygotes had spontaneous fractures and a high frequency of premature lethality at around 2 mo of age. Microcomputed tomography analysis and histomorphometric data revealed a dramatic reduction of both trabecular and cortical bone mass in homozygous mutants. Bone formation in homozygotes was severely impaired, but no obvious phenotypic change was observed in mice heterozygous for the conditional deletion. In vitro studies showed that Wls-deficient osteoblasts had a defect in differentiation and mineralization, with significant reductions in the expression of key osteoblast differentiation regulators. In summary, these results reveal a surprising and crucial role of osteoblast-secreted Wnt ligands in bone-mass accrual. PMID:22745162

  2. Wntless functions in mature osteoblasts to regulate bone mass.

    PubMed

    Zhong, Zhendong; Zylstra-Diegel, Cassandra R; Schumacher, Cassie A; Baker, Jacob J; Carpenter, April C; Rao, Sujata; Yao, Wei; Guan, Min; Helms, Jill A; Lane, Nancy E; Lang, Richard A; Williams, Bart O

    2012-08-14

    Recent genome-wide association studies of individuals of Asian and European descent have found that SNPs located within the genomic region (1p31.3) encoding the Wntless (Wls)/Gpr177 protein are associated significantly with reduced bone mineral density. Wls/Gpr177 is a newly identified chaperone protein that specifically escorts Wnt ligands for secretion. Given the strong functional association between the Wnt signaling pathways and bone development and homeostasis, we generated osteoblast-specific Wls-deficient (Ocn-Cre;Wls-flox) mice. Homozygous conditional knockout animals were born at a normal Mendelian frequency. Whole-body dual-energy X-ray absorptiometry scanning revealed that bone-mass accrual was significantly inhibited in homozygotes as early as 20 d of age. These homozygotes had spontaneous fractures and a high frequency of premature lethality at around 2 mo of age. Microcomputed tomography analysis and histomorphometric data revealed a dramatic reduction of both trabecular and cortical bone mass in homozygous mutants. Bone formation in homozygotes was severely impaired, but no obvious phenotypic change was observed in mice heterozygous for the conditional deletion. In vitro studies showed that Wls-deficient osteoblasts had a defect in differentiation and mineralization, with significant reductions in the expression of key osteoblast differentiation regulators. In summary, these results reveal a surprising and crucial role of osteoblast-secreted Wnt ligands in bone-mass accrual.

  3. Galectin-3 Inhibits Osteoblast Differentiation through Notch Signaling12

    PubMed Central

    Nakajima, Kosei; Kho, Dhong Hyo; Yanagawa, Takashi; Harazono, Yosuke; Gao, Xiaoge; Hogan, Victor; Raz, Avraham

    2014-01-01

    Patients with bone cancer metastasis suffer from unbearable pain and bone fractures due to bone remodeling. This is caused by tumor cells that disturb the bone microenvironment. Here, we have investigated the role of tumor-secreted sugar-binding protein, i.e., galectin-3, on osteoblast differentiation and report that it downregulates the expression of osteoblast differentiation markers, e.g., RUNX2, SP7, ALPL, COL1A1, IBSP, and BGLAP, of treated human fetal osteoblast (hFOB) cells. Co-culturing of hFOB cells with human breast cancer BT-549 and prostate cancer LNCaP cells harboring galectin-3 has resulted in inhibition of osteoblast differentiation by the secreted galectin-3 into culture medium. The inhibitory effect of galectin-3 was found to be through its binding to Notch1 in a sugar-dependent manner that has led to accelerated Notch1 cleavage and activation of Notch signaling. Taken together, our findings show that soluble galectin-3 in the bone microenvironment niche regulates bone remodeling through Notch signaling, suggesting a novel bone metastasis therapeutic target. PMID:25425968

  4. Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

    PubMed

    Ma, Yun-Yun; Sun, Lin; Chen, Xiu-Juan; Wang, Na; Yi, Peng-Fei; Song, Min; Zhang, Bo; Wang, Yu-Zhong; Liang, Qiu-Hua

    2016-01-01

    Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification. PMID:27589055

  5. Extracellular glutamate alters mature osteoclast and osteoblast functions.

    PubMed

    Seidlitz, Eric P; Sharma, Mohit K; Singh, Gurmit

    2010-09-01

    Glutamatergic intercellular communication is involved in many aspects of metabolic homeostasis in normal bone. In bone metastasis, the balance between bone formation and degradation is disrupted. Although the responsible mechanisms are not clear, we have previously identified that cancer cell lines used in bone tumour models secrete glutamate, suggesting that tumour-derived glutamate may disrupt sensitive signalling systems in bone. This study examines the role of glutamate in mature osteoclastic bone resorption, osteoblast differentiation, and bone nodule formation. Glutamate was found to have no effect on the survival or activity of mature osteoclasts, although glutamate transporter inhibition and receptor blockade increased the number of bone resorption pits. Furthermore, transporter inhibition increased the area of resorbed bone while significantly decreasing the number of osteoclasts. Alkaline phosphatase activity and extracellular matrix mineralization were used as measurements of osteoblast differentiation. Glutamate significantly increased osteoblast differentiation and mineralization, but transport inhibitors had no effect. These studies support earlier findings suggesting that glutamate may be more important for osteoclastogenesis than for osteoclast proliferation or functions. Since glutamate is capable of changing the differentiation and activities of both osteoclast and osteoblast cell types in bone, it is reasonable to postulate that tumour-derived glutamate may impact bone homeostasis in bone metastasis.

  6. Osteoblastic meningioma with turtle shell: Different entity from calcified meningioma

    PubMed Central

    Salunke, Pravin; Aggarwal, Ashish; Futane, Sameer; Nada, Ritambhara; Gochhait, Debasis

    2016-01-01

    Bone formation within meningioma is secondary to metaplasia of the meningothelial cells into osteoblastic cells. This needs to be differentiated form the commonly seen calcification. We describe a rare case of osteobalstic meningioma in which bony trabeculae were seen within meningothelial cells. PMID:27695563

  7. Nanostructured magnesium has fewer detrimental effects on osteoblast function

    PubMed Central

    Weng, Lucy; Webster, Thomas J

    2013-01-01

    Efforts have been made recently to implement nanoscale surface features on magnesium, a biodegradable metal, to increase bone formation. Compared with normal magnesium, nanostructured magnesium has unique characteristics, including increased grain boundary properties, surface to volume ratio, surface roughness, and surface energy, which may influence the initial adsorption of proteins known to promote the function of osteoblasts (bone-forming cells). Previous studies have shown that one way to increase nanosurface roughness on magnesium is to soak the metal in NaOH. However, it has not been determined if degradation of magnesium is altered by creating nanoscale features on its surface to influence osteoblast density. The aim of the present in vitro study was to determine the influence of degradation of nanostructured magnesium, created by soaking in NaOH, on osteoblast density. Our results showed a less detrimental effect of magnesium degradation on osteoblast density when magnesium was treated with NaOH to create nanoscale surface features. The detrimental degradation products of magnesium are of significant concern when considering use of magnesium as an orthopedic implant material, and this study identified a surface treatment, ie, soaking in NaOH to create nanoscale features for magnesium that can improve its use in numerous orthopedic applications. PMID:23674891

  8. Jagged1 is essential for osteoblast development during maxillary ossification.

    PubMed

    Hill, Cynthia R; Yuasa, Masato; Schoenecker, Jonathan; Goudy, Steven L

    2014-05-01

    Maxillary hypoplasia occurs due to insufficient maxillary intramembranous ossification, leading to poor dental occlusion, respiratory obstruction and cosmetic deformities. Conditional deletion of Jagged1 (Jag1) in cranial neural crest (CNC) cells using Wnt1-cre; Jagged1(f/f) (Jag1CKO) led to maxillary hypoplasia characterized by intrinsic differences in bone morphology and density using μCT evaluation. Jag1CKO maxillas revealed altered collagen deposition, delayed ossification, and reduced expression of early and late determinants of osteoblast development during maxillary ossification. In vitro bone cultures on Jag1CKO mouse embryonic maxillary mesenchymal (MEMM) cells demonstrated decreased mineralization that was also associated with diminished induction of osteoblast determinants. BMP receptor expression was dysregulated in the Jag1CKO MEMM cells suggesting that these cells were unable to respond to BMP-induced differentiation. JAG1-Fc rescued in vitro mineralization and osteoblast gene expression changes. These data suggest that JAG1 signaling in CNC-derived MEMM cells is required for osteoblast development and differentiation during maxillary ossification. PMID:24491691

  9. Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells

    PubMed Central

    Chen, Xiu-Juan; Wang, Na; Yi, Peng-Fei; Song, Min; Zhang, Bo; Wang, Yu-Zhong; Liang, Qiu-Hua

    2016-01-01

    Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification. PMID:27589055

  10. Osteoblastic meningioma with turtle shell: Different entity from calcified meningioma

    PubMed Central

    Salunke, Pravin; Aggarwal, Ashish; Futane, Sameer; Nada, Ritambhara; Gochhait, Debasis

    2016-01-01

    Bone formation within meningioma is secondary to metaplasia of the meningothelial cells into osteoblastic cells. This needs to be differentiated form the commonly seen calcification. We describe a rare case of osteobalstic meningioma in which bony trabeculae were seen within meningothelial cells.

  11. Specific proteins mediate enhanced osteoblast adhesion on nanophase ceramics.

    PubMed

    Webster, T J; Ergun, C; Doremus, R H; Siegel, R W; Bizios, R

    2000-09-01

    Osteoblast, fibroblast, and endothelial cell adhesion on nanophase (that is, materials with grain sizes less than 100 nm) alumina, titania, and hydroxyapatite (HA) was investigated using in vitro cellular models. Osteoblast adhesion was significantly (p < 0.01) greater after 4 h on nanophase alumina, titania, and HA than it was on conventional formulations of the same ceramics. In contrast, compared to conventional alumina, titania, and HA, after 4 h fibroblast adhesion was significantly (p < 0.01) less on nanophase ceramics. Examination of the underlying mechanism(s) of cell adhesion on nanophase ceramics revealed that these ceramics adsorbed significantly (p < 0.01) greater quantities of vitronectin, which, subsequently, may have contributed to the observed select enhanced adhesion of osteoblasts. Select enhanced osteoblast adhesion was independent of surface chemistry and material phase but was dependent on the surface topography (specifically on grain and pore size) of nanophase ceramics. The capability of synthesizing and processing nanomaterials with tailored (through, for example, specific grain and pore size) structures and topographies to control select subsequent cell functions provides the possibility of designing the novel proactive biomaterials (that is, materials that elicit specific, timely, and desirable responses from surrounding cells and tissues) necessary for improved implant efficacy.

  12. Sulfuretin induces osteoblast differentiation through activation of TGF-β signaling.

    PubMed

    Song, No-Joon; Kwon, So-Mi; Kim, Suji; Yoon, Hyang-Jin; Seo, Cho-Rong; Jang, Byunghyun; Chang, Seo-Hyuk; Ku, Jin-Mo; Lee, Jeong-Soo; Park, Ki-Moon; Hong, Joung-Woo; Kim, Geun Hyung; Park, Kye Won

    2015-12-01

    The identification and examination of potential determinants controlling the progression of cell fate toward osteoblasts can be intriguing subjects. In this study, the effects of sulfuretin, a major compound isolated from Rhus verniciflua Stokes, on osteoblast differentiation were investigated. Treatments of sulfuretin induced alkaline phosphatase (ALP) activity in mesenchymal C3H10T1/2 cells and mineralization in preosteoblast MC3T3-E1 cells. Pro-osteogenic effects of sulfuretin were consistently observed in freshly isolated primary bone marrow cells. In mechanical studies, sulfuretin specifically induced expression of TGF-β target genes, such as SMAD7 and PAI-1, but not other signaling pathway-related genes. Similar to the results of gene expression analysis, reporter assays further demonstrated TGF-β-specific induction by sulfuretin. Furthermore, disruption of TGF-β signaling using treatment with TGF-β-specific inhibitor, SB-431542, and introduction of SMAD2/3 small interfering RNA impaired the effects of sulfuretin in inducing ALP activity and expression of ALP mRNA. Together, these data indicate that the pro-osteogenic effects of sulfuretin are mediated through activation of TGF-β signaling, further supporting the potential of sulfuretin in the prevention of bone-related diseases such as bone fracture and osteoporosis.

  13. Enhanced osteoblast-like cell adhesion and proliferation using sulfonate-bearing polymeric scaffolds.

    PubMed

    Chaterji, Somali; Gemeinhart, Richard A

    2007-12-15

    Orthopedic malfunction, degeneration, or damage remains a serious healthcare issue despite advances in medical technology. Proactive extracellular matrix (ECM)-mimetic scaffolds are being researched to orchestrate the activation of diverse osteogenic signaling cascades, facilitating osteointegration. We hypothesized that sulfonated functionalities incorporated into synthetic hydrogels would simulate anionic, sulfate-bearing proteoglycans, abundant in the ECM. Using this rationale, we successfully developed differentially sulfonated hydrogels, polymerizing a range of sulfopropyl acrylate potassium-acrylamide (SPAK-AM) mole ratios as monomer feeds under room temperature conditions. For anchorage-dependent cells, such as osteoblasts, adhesion is a critical prerequisite for subsequent osteointegration and cell specialization. The introduction of the sulfonated monomer, SPAK, resulted in favorable uptake of serum proteins with proportional increase in adhesion and proliferation rates of model cell lines, which included NIH/3T3 fibroblasts, MG-63 osteoblasts, and MC3T3-E1 subclone 4 preosteoblasts. In fact, higher proportions of sulfonate content (pSPAK75, pSPAK100) exhibited comparable or even higher degrees of adhesion and proliferation, relative to commercial grade tissue culture polystyrene in vitro. These results indicate promising potentials of sulfonated ECM-mimetic hydrogels as potential osteogenic tissue engineering scaffolds. PMID:17584889

  14. Characterization of Scaffold Carriers for BMP9-Transduced Osteoblastic Progenitor Cells in Bone Regeneration

    PubMed Central

    Shui, Wei; Zhang, Wenwen; Yin, Liangjun; Nan, Guoxin; Liao, Zhan; Zhang, Hongmei; Wang, Ning; Wu, Ningning; Chen, Xian; Wen, Sheng; He, Yunfeng; Deng, Fang; Zhang, Junhui; Luu, Hue H.; Shi, Lewis L; Hu, Zhenming; Haydon, Rex C.; Mok, James; He, Tong-Chuan

    2015-01-01

    Successful bone tissue engineering at least requires sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We have demonstrated that BMP9 is one of the most potent factors in inducing osteogenic differentiation of mesenchymal progenitors. To facilitate the potential use of cell-based BMP9 gene therapy for bone regeneration, we characterize the in vivo osteoconductive activities and bone regeneration potential of three clinically-used scaffold materials, type I collagen sponge, hydroxyapatite-tricalcium phosphate (HA-TCP) and demineralized bone matrix (DBM), using BMP9-expressing C2C12 osteoblastic progenitor cells. We find that recombinant adenovirus-mediated BMP9 expression effectively induces osteogenic differentiation in C2C12 cells. Although direct subcutaneous injection of BMP9-transduced C2C12 cells forms ectopic bony masses, subcutaneous implantation of BMP9-expressing C2C12 cells with collagen sponge or HA-TCP scaffold yields the most robust and mature cancellous bone formation, whereas the DBM carrier group forms no or minimal bone masses. Our results suggest that collagen sponge and HA-TCP scaffold carriers may provide more cell-friendly environment to support the survival, propagation, and ultimately differentiation of BMP9-expressing progenitor cells. This line of investigation should provide important experimental evidence for further pre-clinical studies in BMP9-mediated cell based approach to bone tissue engineering. PMID:24133046

  15. Improving effects of chitosan nanofiber scaffolds on osteoblast proliferation and maturation

    PubMed Central

    Ho, Ming-Hua; Liao, Mei-Hsiu; Lin, Yi-Ling; Lai, Chien-Hao; Lin, Pei-I; Chen, Ruei-Ming

    2014-01-01

    Osteoblast maturation plays a key role in regulating osteogenesis. Electrospun nanofibrous products were reported to possess a high surface area and porosity. In this study, we developed chitosan nanofibers and examined the effects of nanofibrous scaffolds on osteoblast maturation and the possible mechanisms. Macro- and micro observations of the chitosan nanofibers revealed that these nanoproducts had a flat surface and well-distributed fibers with nanoscale diameters. Mouse osteoblasts were able to attach onto the chitosan nanofiber scaffolds, and the scaffolds degraded in a time-dependent manner. Analysis by scanning electron microscopy further showed mouse osteoblasts adhered onto the scaffolds along the nanofibers, and cell–cell communication was also detected. Mouse osteoblasts grew much better on chitosan nanofiber scaffolds than on chitosan films. In addition, human osteoblasts were able to adhere and grow on the chitosan nanofiber scaffolds. Interestingly, culturing human osteoblasts on chitosan nanofiber scaffolds time-dependently increased DNA replication and cell proliferation. In parallel, administration of human osteoblasts onto chitosan nanofibers significantly induced osteopontin, osteocalcin, and alkaline phosphatase (ALP) messenger (m)RNA expression. As to the mechanism, chitosan nanofibers triggered runt-related transcription factor 2 mRNA and protein syntheses. Consequently, results of ALP-, alizarin red-, and von Kossa-staining analyses showed that chitosan nanofibers improved osteoblast mineralization. Taken together, results of this study demonstrate that chitosan nanofibers can stimulate osteoblast proliferation and maturation via runt-related transcription factor 2-mediated regulation of osteoblast-associated osteopontin, osteocalcin, and ALP gene expression. PMID:25246786

  16. Osteoblast maturation occurs in overlapping proximal-distal compartments during fin regeneration in zebrafish.

    PubMed

    Brown, Andrew M; Fisher, Shannon; Iovine, M Kathryn

    2009-11-01

    During fin regeneration, osteoblasts must continually differentiate for outgrowth of the bony fin rays. Bone maturity increases in a distal-proximal manner, and osteoblast maturation can be detected similarly when following gene expression. We find that early markers for osteoblast differentiation are expressed in a discrete domain at the distal end of the fin, just proximal to the adjacent germinal compartment of dividing cells. Matrix genes, required at later stages developmentally, are expressed in a population of cells proximally to the early genes. A marker for mature osteoblasts is expressed in cells further proximal. These domains of gene expression are partially overlapping, perhaps revealing additional levels of osteoblast maturity. We suggest a model for growth where new cells are continually added to the distal-most osteoblast compartment, while osteoblasts in more proximal locations differentiate, thus translating developmental time to location on the proximal-distal axis. PMID:19842180

  17. A functional polyester carrying free hydroxyl groups promotes the mineralization of osteoblast and human mesenchymal stem cell extracellular matrix.

    PubMed

    Bi, Xiaoping; You, Zhengwei; Gao, Jin; Fan, Xianqun; Wang, Yadong

    2014-06-01

    Functional groups can control biointerfaces and provide a simple way to make therapeutic materials. We recently reported the design and synthesis of poly(sebacoyl diglyceride) (PSeD) carrying a free hydroxyl group in its repeating unit. This paper examines the use of this polymer to promote biomineralization for application in bone tissue engineering. PSeD promoted more mineralization of extracellular matrix secreted by human mesenchymal stem cells and rat osteoblasts than poly(lactic-co-glycolic acid) (PLGA), which is currently widely used in bone tissue engineering. PSeD showed in vitro osteocompatibility and in vivo biocompatibility that matched or surpassed that of PLGA, as well as supported the attachment, proliferation and differentiation of rat osteoblasts and human mesenchymal stem cells. This demonstrates the potential of PSeD for use in bone regeneration. PMID:24560799

  18. Relaxin augments BMP-2-induced osteoblast differentiation and bone formation.

    PubMed

    Moon, Jung-Sun; Kim, Sun-Hun; Oh, Sin-Hye; Jeong, Yong-Wook; Kang, Jee-Hae; Park, Jong-Chun; Son, Hye-Ju; Bae, Suk; Park, Byung-Il; Kim, Min-Seok; Koh, Jeong-Tae; Ko, Hyun-Mi

    2014-07-01

    Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP-2)-induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT-PCR and Western blot during BMP-2-induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP-2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro-computed tomography (µCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation-related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP-2. In vitro, Rln augmented BMP-2-induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP-2-induced bone formation in a dose-dependent manner. Interestingly, Rln synergistically increased and sustained BMP-2-induced Smad, p38, and transforming growth factor-β activated kinase (TAK) 1 phosphorylation. BMP-2-induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP-2-induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP-2-induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone

  19. Megakaryocytes are mechanically responsive and influence osteoblast proliferation and differentiation.

    PubMed

    Soves, Constance P; Miller, Joshua D; Begun, Dana L; Taichman, Russell S; Hankenson, Kurt D; Goldstein, Steven A

    2014-09-01

    Maintenance of bone mass and geometry is influenced by mechanical stimuli. Paradigms suggest that osteocytes embedded within the mineralized matrix and osteoblasts on the bone surfaces are the primary responders to physical forces. However, other cells within the bone marrow cavity, such as megakaryocytes (MKs), are also subject to mechanical forces. Recent studies have highlighted the potent effects of MKs on osteoblast proliferation as well as bone formation in vivo. We hypothesize that MKs are capable of responding to physical forces and that the interactions between these cells and osteoblasts can be influenced by mechanical stimulation. In this study, we demonstrate that two MK cell lines respond to fluid shear stress in culture. Furthermore, using laser capture microdissection, we isolated MKs from histologic sections of murine tibiae that were exposed to compressive loads in vivo. C-fos, a transcription factor shown to be upregulated in response to load in various tissue types, was increased in MKs from loaded relative to non-loaded limbs at a level comparable to that of osteocytes from the same limbs. We also developed a co-culture system to address whether mechanical stimulation of MKs in culture would impact osteoblast proliferation and differentiation. The presence of MKs in co-culture, but not conditioned media, had dramatic effects on proliferation of preosteoblast MC3T3-E1 cells in culture. Our data suggests a minimal decrease in proliferation as well as an increase in mineralization capacity of osteoblasts co-cultured with MKs exposed to shear compared to co-cultures with unstimulated MKs. PMID:24882736

  20. Effects of cefuroxime on human osteoblasts in vitro.

    PubMed

    Salzmann, G M; Naal, F D; von Knoch, F; Tuebel, J; Gradinger, R; Imhoff, A B; Schauwecker, J

    2007-08-01

    The local application of antibiotics in bone cement achieves high local effective antibiotic concentrations. Cefuroxime is widely used for antibiotic prophylaxis in orthopedic surgery, and several reports highlighted a beneficial outcome if cefuroxime-impregnated bone cement was used, but there is a lack of information of direct cefuroxime effects on human bone cells. We, therefore, cultured osteoblasts, previously derived from human trabecular bone specimens and used as a cell-pool further on, with different concentrations of cefuroxime (0-1000 microg/mL) for 24, 48, or 72 h. For reversibility testing, osteoblasts were cultivated for 24 h with cefuroxime followed by 48 h without antibiotics. Cell proliferation (MTT), cytotoxicity (lactate dehydrogenase (LDH)-activity), cell metabolism (alkaline phosphatase (ALP)-activity), and extracellular matrix calcification (Alizarin staining) were assessed after antibiotic treatment. Cefuroxime concentrations of 25-100 microg/mL had little or no effect on cellular proliferation. Proliferation was significantly stimulated at 250 and 1000 microg/mL at each time. LDH-activity significantly increased at the highest concentration of 1000 microg/mL at 72 h. ALP-activity first increased at lower concentrations and then significantly decreased at 1000 microg/mL at 48 and 72 h. Similar to ALP-activity, calcification increased at lower concentrations and was not detectable at 1000 microg/mL. All revealed effects at 24 h were at least partially reversible. In the present study, we demonstrated that cefuroxime at lower concentrations had no inhibiting effects on human osteoblasts. In contrast, higher concentrations significantly altered osteoblastic function. When administered locally in total joint arthroplasty, for example, in antibiotic-impregnated bone cement, cefuroxime might critically impair osteoblastic function and periprosthetic bone metabolism.

  1. RXP-E: A CX43-BINDING PEPTIDE THAT PREVENTS ACTION POTENTIAL PROPAGATION BLOCK

    PubMed Central

    Lewandowski, Rebecca; Procida, Kristina; Vaidyanathan, Ravi; Coombs, Wanda; Jalife, Jose; Nielsen, Morten S.; Taffet, Steven M.; Delmar, Mario

    2009-01-01

    Gap junctions (GJs) provide a low-resistance pathway for cardiac electrical propagation. The role of GJ regulation in arrhythmia is unclear, partly due to limited availability of pharmacological tools. Recently, we showed that a peptide called “RXP-E” binds to the carboxyl terminal of connexin43 (Cx43) and prevents chemically-induced uncoupling in Cx43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac Cx43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes (NRVM’s). Separately, RXP-E was concatenated to a cytoplasmic transduction peptide for cytoplasmic translocation (CTP-RXP-E). The effect of RXP-E on action potential (AP) propagation was assessed by high resolution optical mapping in monolayers of NRVM’s, containing ~20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, AP propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pHi=6.2) caused a loss of AP propagation in control monolayers; however, propagation was maintained in CTP-RXP-E treated cells, though at a slower rate. Patch clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac Cx43; (2) prevent heptanol and acidification-induced uncoupling of cardiac GJ’s and 3) preserve AP propagation among cardiac myocytes. RXP-E can be used to characterize the role of GJs in the function of multicellular systems, including the heart. PMID:18669919

  2. Co-culture with periodontal ligament stem cells enhanced osteoblastic differentiation of MC3T3-E1 cells and osteoclastic differentiation of RAW264.7 cells

    PubMed Central

    Chen, Shulan; Ye, Xin; Yu, Xinbo; Xu, Quanchen; Pan, Keqing; Lu, Shulai; Yang, Pishan

    2015-01-01

    Objectives: Periodontal ligament stem cells (PDLSCs) are characterized by having multipotential differentiation and immunoregulatory properties, which are the main mechanisms of PDLSCs-mediated periodontal regeneration. Periodontal or bone regeneration requires coordination of osteoblast and osteoclast, however, very little is known about the interactions between PDLSCs and osteoblast-like cells or osteoclast precursors. In this study, the indirect co-culture approach was introduced to preliminarily elucidate the effects of PDLSCs on differentiation of osteoblast-like cells and osteoclast precursors in vitro. Materials and methods: Human PDLSCs were obtained from premolars extracted and their stemness was identified in terms of their colony-forming ability, proliferative capacity, cell surface epitopes and multi-lineage differentiation potentials. A noncontact co-culture system of PDLSCs and preosteoblastic cell line MC3T3-E1 or osteoclast precursor cell line RAW264.7 was established, and osteoblastic differentiation of MC3T3-E1 and osteoclastic differentiation of RAW264.7 were evaluated. Results: PDLSCs exhibited features of mesenchymal stem cells. Further investigation through indirect co-culture system showed that PDLSCs enhanced ALP activity, expressions of ALP, Runx2, BSP, OPN mRNA and BSP, OPN proteins and mineralization matrix deposition in MC3T3-E1. Meanwhile, they improved maturation of osteoclasts and expressions of TRAP, CSTK, TRAF6 mRNA and TRAP, TRAF6 proteins in RAW264.7. Conclusions: PDLSCs stimulates osteoblastic differentiation of osteoblast precursors and osteoclastic differentiation of osteoclast precursors, at least partially, in a paracrine fasion. PMID:26823783

  3. Prostate cancer cells preferentially home to osteoblast-rich areas in the early stages of bone metastasis: evidence from in vivo models.

    PubMed

    Wang, Ning; Docherty, Freyja E; Brown, Hannah K; Reeves, Kimberley J; Fowles, Anne C M; Ottewell, Penelope D; Dear, T Neil; Holen, Ingunn; Croucher, Peter I; Eaton, Colby L

    2014-12-01

    It has been suggested that metastasis-initiating cells gain a foothold in bone by homing to a metastastatic microenvironment (or "niche"). Whereas the precise nature of this niche remains to be established, it is likely to contain bone cell populations including osteoblasts and osteoclasts. In the mouse tibia, the distribution of osteoblasts on endocortical bone surfaces is non-uniform, and we hypothesize that studying co-localization of individual tumor cells with resident cell populations will reveal the identity of critical cellular components of the niche. In this study, we have mapped the distribution of three human prostate cancer cell lines (PC3-NW1, LN-CaP, and C4 2B4) colonizing the tibiae of athymic mice following intracardiac injection and evaluated their interaction with potential metastatic niches. Prostate cancer cells labeled with the fluorescent cell membrane dye (Vybrant DiD) were found by two-photon microscopy to be engrafted in the tibiae in close proximity (∼40 µm) to bone surfaces and 70% more cancer cells were detected in the lateral compared to the medial endocortical bone regions. This was associated with a 5-fold higher number of osteoblasts and 7-fold higher bone formation rate on the lateral endocortical bone surface compared to the medial side. By disrupting cellular interactions mediated by the chemokine (C-X-C motif) receptor 4 (CXCR4)/chemokine ligand 12 (CXCL12) axis with the CXCR4 inhibitor AMD3100, the preferential homing pattern of prostate cancer cells to osteoblast-rich bone surfaces was disrupted. In this study, we map the location of prostate cancer cells that home to endocortical regions in bone and our data demonstrate that homing of prostate cancer cells is associated with the presence and activity of osteoblast lineage cells, and suggest that therapies targeting osteoblast niches should be considered to prevent development of incurable prostate cancer bone metastases.

  4. Fucoidan promotes osteoblast differentiation via JNK- and ERK-dependent BMP2-Smad 1/5/8 signaling in human mesenchymal stem cells.

    PubMed

    Kim, Beom Su; Kang, Hyo-Jin; Park, Ji-Yun; Lee, Jun

    2015-01-01

    Fucoidan has attracted attention as a potential drug because of its biological activities, which include osteogenesis. However, the molecular mechanisms involved in the osteogenic activity of fucoidan in human alveolar bone marrow-derived mesenchymal stem cells (hABM-MSCs) remain largely unknown. We investigated the action of fucoidan on osteoblast differentiation in hABM-MSCs and its impact on signaling pathways. Its effect on proliferation was determined using the crystal violet staining assay. Osteoblast differentiation was evaluated based on alkaline phosphatase (ALP) activity and the mRNA expression of multiple osteoblast markers. Calcium accumulation was determined by Alizarin red S staining. We found that fucoidan induced hABM-MSC proliferation. It also significantly increased ALP activity, calcium accumulation and the expression of osteoblast-specific genes, such as ALP, runt-related transcription factor 2, type I collagen-α 1 and osteocalcin. Moreover, fucoidan induced the expression of bone morphogenetic protein 2 (BMP2) and stimulated the activation of extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase by increasing phosphorylation. However, the effect of fucoidan on osteogenic differentiation was inhibited by specific inhibitors of ERK (PD98059) and JNK (SP600125) but not p38 (SB203580). Fucoidan enhanced BMP2 expression and Smad 1/5/8, ERK and JNK phosphorylation. Moreover, the effect of fucoidan on osteoblast differentiation was diminished by BMP2 knockdown. These results indicate that fucoidan induces osteoblast differentiation through BMP2-Smad 1/5/8 signaling by activating ERK and JNK, elucidating the molecular basis of the osteogenic effects of fucoidan in hABM-MSCs.

  5. Evidence of vitamin D and interferon-β cross-talk in human osteoblasts with 1α,25-dihydroxyvitamin D3 being dominant over interferon-β in stimulating mineralization.

    PubMed

    Woeckel, V J; Koedam, M; van de Peppel, J; Chiba, H; van der Eerden, B C J; van Leeuwen, J P T M

    2012-09-01

    It is well established that 1α-25-dihydroxyvitamin D3 (1,25D3) regulates osteoblast function and stimulates mineralization by human osteoblasts. The aim of this study was to identify processes underlying the 1,25D3 effects on mineralization. We started with gene expression profiling analyses of differentiating human pre-osteoblast treated with 1,25D3. Bioinformatic analyses showed interferon-related and -regulated genes (ISG) to be overrepresented in the set of 1,25D3-regulated genes. 1,25D3 down-regulated ISGs predominantly during the pre-mineralization period. This pointed to an interaction between the vitamin D and IFN signaling cascades in the regulation of osteoblast function. Separately, 1,25D3 enhances while IFNβ inhibits mineralization. Treatment of human osteoblasts with 1,25D3 and IFNβ showed that 1,25D3 completely overrules the IFNβ inhibition of mineralization. This was supported by analyses of extracellular matrix gene expression, showing a dominant effect of 1,25D3 over the inhibitory effect of IFNβ. We identified processes shared by IFNβ- and 1,25D3-mediated signaling by performing gene expression profiling during early osteoblast differentiation. Bioinformatic analyses revealed that genes being correlated or anti-correlated with interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were associated with osteoblast proliferation. In conclusion, the current study demonstrates a cross talk between 1,25D3 and IFNβ in osteoblast differentiation and bone formation/mineralization. The interaction is complex and depends on the process but importantly, 1,25D3 stimulation of mineralization is dominant over the inhibitory effect of IFNβ. These observations are of potential clinical relevance considering the impact of the immune system on bone metabolism in conditions such as rheumatoid arthritis.

  6. Time- and dose-related interactions between glucocorticoid and cyclic adenosine 3',5'-monophosphate on CCAAT/enhancer-binding protein-dependent insulin-like growth factor I expression by osteoblasts

    NASA Technical Reports Server (NTRS)

    McCarthy, T. L.; Ji, C.; Chen, Y.; Kim, K.; Centrella, M.

    2000-01-01

    Glucocorticoid has complex effects on osteoblasts. Several of these changes appear to be related to steroid concentration, duration of exposure, or specific effects on growth factor expression or activity within bone. One important bone growth factor, insulin-like growth factor I (IGF-I), is induced in osteoblasts by hormones such as PGE2 that increase intracellular cAMP levels. In this way, PGE2 activates transcription factor CCAAT/enhancer-binding protein-delta (C/EBPdelta) and enhances its binding to a specific control element found in exon 1 in the IGF-I gene. Our current studies show that preexposure to glucocorticoid enhanced C/EBPdelta and C/EBPbeta expression by osteoblasts and thereby potentiated IGF-I gene promoter activation in response to PGE2. Importantly, this directly contrasts with inhibitory effects on IGF-I expression that result from sustained or pharmacologically high levels of glucocorticoid exposure. Consistent with the stimulatory effect of IGF-I on bone protein synthesis, pretreatment with glucocorticoid sensitized osteoblasts to PGE2, and in this context significantly enhanced new collagen and noncollagen protein synthesis. Therefore, pharmacological levels of glucocorticoid may reduce IGF-I expression by osteoblasts and cause osteopenic disease, whereas physiological transient increases in glucocorticoid may permit or amplify the effectiveness of hormones that regulate skeletal tissue integrity. These events appear to converge on the important role of C/EBPdelta and C/EBPbeta on IGF-I expression by osteoblasts.

  7. Decreased oxygen tension lowers reactive oxygen species and apoptosis and inhibits osteoblast matrix mineralization through changes in early osteoblast differentiation.

    PubMed

    Nicolaije, Claudia; Koedam, Marijke; van Leeuwen, Johannes P T M

    2012-04-01

    Accumulating data show that oxygen tension can have an important effect on cell function and fate. We used the human pre-osteoblastic cell line SV-HFO, which forms a mineralizing extracellular matrix, to study the effect of low oxygen tension (2%) on osteoblast differentiation and mineralization. Mineralization was significantly reduced by 60-70% under 2% oxygen, which was paralleled by lower intracellular levels of reactive oxygen species (ROS) and apoptosis. Following this reduction in ROS the cells switched to a lower level of protection by down-regulating their antioxidant enzyme expression. The downside of this is that it left the cells more vulnerable to a subsequent oxidative challenge. Total collagen content was reduced in the 2% oxygen cultures and expression of matrix genes and matrix-metabolizing enzymes was significantly affected. Alkaline phosphatase activity and RNA expression as well as RUNX2 expression were significantly reduced under 2% oxygen. Time phase studies showed that high oxygen in the first phase of osteoblast differentiation and prior to mineralization is crucial for optimal differentiation and mineralization. Switching to 2% or 20% oxygen only during mineralization phase did not change the eventual level of mineralization. In conclusion, this study shows the significance of oxygen tension for proper osteoblast differentiation, extra cellular matrix (ECM) formation, and eventual mineralization. We demonstrated that the major impact of oxygen tension is in the early phase of osteoblast differentiation. Low oxygen in this phase leaves the cells in a premature differentiation state that cannot provide the correct signals for matrix maturation and mineralization.

  8. Opposite Function of ERα and ERβ in Controlling 17β-Estradiol-mediated Osteogenesis in Osteoblasts.

    PubMed

    Wang, Yu-Xiang; Li, Min; Zhang, Hong-Qi; Tang, Ming-Xing; Guo, Chao-Feng; Deng, Ang; Chen, Yong; Xiao, Li-Ge

    2016-05-01

    Estrogen receptor plays critical roles in osteogenesis but the underlying mechanism remains unclear. In order to determine the effect of ERα and ERβ on several critical factors in regulating osteogenesis in human osteoblasts. Cell based assy, RT-PCR and immunoblot analyses were used in the research. Both RT-PCR and immunoblot showed that gene expression of OPG, MBP2, TGF-β, RUNX2, IGF-1 was significantly reduced while expression of RANKL was drastically increased after shRNA-based depletion of ERα in MG-63 osteoblasts. Surprisingly, 17β-estradiol (E2) treatment led to remarkably reduced RANKL compared with that in E2 untreated cells. In contrast, ERβ plays an opposite role in regulating gene expression of OPG, MBP2, TGF-β, RUNX2, IGF-1 and RANKL. However, double depletion of ERα and ERβ could not rescue the gene expression of these factors in vitro. Our results provide a novel mechanism of estrogen receptor in controlling osteogenesis in human cells as well as a potential clinic therapeutic target in human osteoporosis. PMID:27664484

  9. The role of biosilica in the osteoprotegerin/RANKL ratio in human osteoblast-like cells.

    PubMed

    Wiens, Matthias; Wang, Xiaohong; Schröder, Heinz C; Kolb, Ute; Schlossmacher, Ute; Ushijima, Hiroshi; Müller, Werner E G

    2010-10-01

    Earlier studies have demonstrated that biosilica, synthesized by the enzyme silicatein, induces hydroxyapatite formation in osteoblast-like SaOS-2 cells. Here we study the effect of biosilica on the expressions of osteoprotegerin [OPG] and the receptor activator for NF-kappaB ligand [RANKL] in the SaOS-2 cell model. We show that during growth of SaOS-2 cells on biosiliceous matrices hydroxyapatite formation is induced, while syntheses of cartilaginous proteoglycans and sulfated glycosaminoglycans are down-regulated. Furthermore, quantitative real-time RT-PCR analysis revealed a strong time-depended increase in expression of OPG in biosilica exposed SaOS-2 cells while the steady-state expression level of RANKL remained unchanged. These results have been corroborated on the protein level by ELISA assays. Therefore, we propose that biosilica stimulated OPG synthesis in osteoblast-like cells counteracts those pathways that control RANKL expression and function (e.g. maturation of pre-osteoclasts and activation of osteoclasts). Hence, the data obtained in the present study reveal the considerable biomedical potential of biosilica for treatment and prophylaxis of osteoporotic disorders.

  10. Nuclear morphometric analysis of osteoblast precursor cells in periodontal ligament, SL-3 rats

    NASA Technical Reports Server (NTRS)

    Roberts, W. E.; Fielder, P. J.; Rosenoer, L. M.; Maese, A. C.; Gonsalves, M. R.; Morey, E. R.; Morey-Holton, E. R. (Principal Investigator)

    1987-01-01

    Five small (55 days old, 196 +/- 5 g) (mean +/- SE) and five large (83 days old, 382 +/- 4 g) Sprague-Dawley strain, specific pathogen-free rats were exposed to a 7-day spaceflight and 12-h postflight recovery period. As measured in 3-micron sections, periodontal ligament (PDL) fibroblastlike cells were classified according to nuclear size: A + A' (40-79), B (80-119), C (120-169), and D (greater than or equal to 170 microns 3). Since the histogenesis sequence is A----A'----C----D----osteoblast, the relative incidence of A + A' to C + D is an osteogenic index. No difference in A + A' or C + D cells in small rats may reflect partial recovery of preosteoblast formation (A----C) during the 12-h postflight period. Large flight rats demonstrated increased numbers of A + A', indicating an inhibition of preosteoblast formation (A----C). At least in the older group, a 7-day flight is adequate to reduce PDL osteogenic potential (inhibition in PDL osteoblast differentiation and/or specific attrition of C + D cells) that does not recover by 12-h postflight.

  11. Effects of Chitin Whiskers on Physical Properties and Osteoblast Culture of Alginate Based Nanocomposite Hydrogels.

    PubMed

    Huang, Yao; Yao, Mengyu; Zheng, Xing; Liang, Xichao; Su, Xiaojuan; Zhang, Yu; Lu, Ang; Zhang, Lina

    2015-11-01

    Novel nanocomposite hydrogels composed of polyelectrolytes alginate and chitin whiskers with biocompatibility were successfully fabricated based on the pH-induced charge shifting behavior of chitin whiskers. The chitin whiskers with mean length and width of 300 and 20 nm were uniformly dispersed in negatively charged sodium alginate aqueous solution, leading to the formation of the homogeneous nanocomposite hydrogels. The experimental results indicated that their mechanical properties were significantly improved compared to alginate hydrogel and the swelling trends were inhibited as a result of the strong electrostatic interactions between the chitin whiskers and alginate. The nanocomposite hydrogels exhibited certain crystallinity and hierarchical structure with nanoscale chitin whiskers, similar to the structure of the native extracellular matrix. Moreover, the nanocomposite hydrogels were successfully applied as bone scaffolds for MC3T3-E1 osteoblast cells, showing their excellent biocompatibility and low cytotoxicity. The results of fluorescent micrographs and scanning electronic microscope (SEM) images revealed that the addition of chitin whiskers into the nanocomposite hydrogels markedly promoted the cell adhesion and proliferation of the osteoblast cells. The biocompatible nanocomposite hydrogels have potential application in bone tissue engineering. PMID:26393272

  12. Effects of Chitin Whiskers on Physical Properties and Osteoblast Culture of Alginate Based Nanocomposite Hydrogels.

    PubMed

    Huang, Yao; Yao, Mengyu; Zheng, Xing; Liang, Xichao; Su, Xiaojuan; Zhang, Yu; Lu, Ang; Zhang, Lina

    2015-11-01

    Novel nanocomposite hydrogels composed of polyelectrolytes alginate and chitin whiskers with biocompatibility were successfully fabricated based on the pH-induced charge shifting behavior of chitin whiskers. The chitin whiskers with mean length and width of 300 and 20 nm were uniformly dispersed in negatively charged sodium alginate aqueous solution, leading to the formation of the homogeneous nanocomposite hydrogels. The experimental results indicated that their mechanical properties were significantly improved compared to alginate hydrogel and the swelling trends were inhibited as a result of the strong electrostatic interactions between the chitin whiskers and alginate. The nanocomposite hydrogels exhibited certain crystallinity and hierarchical structure with nanoscale chitin whiskers, similar to the structure of the native extracellular matrix. Moreover, the nanocomposite hydrogels were successfully applied as bone scaffolds for MC3T3-E1 osteoblast cells, showing their excellent biocompatibility and low cytotoxicity. The results of fluorescent micrographs and scanning electronic microscope (SEM) images revealed that the addition of chitin whiskers into the nanocomposite hydrogels markedly promoted the cell adhesion and proliferation of the osteoblast cells. The biocompatible nanocomposite hydrogels have potential application in bone tissue engineering.

  13. Osteoblastic cell response on biphasic fluorhydroxyapatite/strontium-substituted hydroxyapatite coatings.

    PubMed

    Yin, Ping; Feng, Fang Fang; Lei, Ting; Zhong, Xiao Huan; Jian, Xin Chun

    2014-03-01

    Fluorhydroxyapatite/strontium-substituted hydroxyapatite (FHA/SrHA) biphasic coatings with F and Sr elements incorporated simultaneously into one coating layer were prepared on titanium substrate via colloidal sol-gel method. The bioactivity of the as-prepared FHA/SrHA biphasic coatings was evaluated in vitro by immersion in simulated body fluid (SBF). All the biphasic coatings exhibited great ability to induce apatite precipitation on their surfaces. In vitro cell responses were evaluated using osteoblast-like MG63 cells in terms of cell proliferation and differentiation (alkaline phosphatase activity and osteocalcin level). The biphasic coatings show significantly positive effects on the viability and functional activity of osteoblastic cells with clear evidence that an optimum SrHA amount dose exists, indicating that the coexistence of FHA and SrHA had a synergistic stimulatory effect. This finding suggests the potential use of this colloidal sol-gel derived FHA/SrHA biphasic coatings for hard tissue applications. PMID:23533202

  14. Polyester copolymer scaffolds enhance expression of bone markers in osteoblast-like cells.

    PubMed

    Idris, Shaza Bushra; Arvidson, Kristina; Plikk, Peter; Ibrahim, Salah; Finne-Wistrand, Anna; Albertsson, Ann-Christine; Bolstad, Anne Isine; Mustafa, Kamal

    2010-08-01

    In tissue engineering, the resorbable aliphatic polyester poly(L-lactide) (PLLA) is used as scaffolds in bone regeneration. Copolymers of poly(L-lactide)-co-(epsilon-caprolactone) [poly(LLA-co-CL)] and poly(L-lactide)-co-(1,5-dioxepan-2-one) [poly(LLA-co-DXO)], with superior mechanical properties to PLLA, have been developed to be used as scaffolds, but the influence on the osteogenic potential is unclear. This in vitro study of test scaffolds of poly(LLA-co-CL) and poly(LLA-co-DXO) using PLLA scaffolds as a control demonstrates the attachment and proliferation of human osteoblast-like cells (HOB) as measured by SEM and a methylthiazol tetrazolium (MTT) colorimetric assay, and the progression of HOB osteogenesis for up to 3 weeks; expressed as synthesis of the osteoblast differentiation markers: collagen type 1 (Col 1), alkaline phosphatase, bone sialoprotein, osteocalcin (OC), osteopontin and runt related gene 2 (Runx2). Surface analysis disclosed excellent surface attachment, spread and penetration of the cells into the pores of the test scaffolds compared to the PLLA. MTT results indicated that test scaffolds enhanced the proliferation of HOBs. Cells grown on the test scaffolds demonstrated higher synthesis of Col 1 and OC and also increased bone markers mRNA expression. Compared to scaffolds of PLLA, the poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds enhanced attachment, proliferation, and expression of osteogenic markers by HOBs in vitro. Therefore, these scaffolds might be appropriate carriers for bone engineering.

  15. Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells.

    PubMed

    Guignandon, Alain; Faure, Céline; Neutelings, Thibaut; Rattner, Aline; Mineur, Pierre; Linossier, Marie-Thérèse; Laroche, Norbert; Lambert, Charles; Deroanne, Christophe; Nusgens, Betty; Demets, René; Colige, Alain; Vico, Laurence

    2014-09-01

    Bone cells exposed to real microgravity display alterations of their cytoskeleton and focal adhesions, two major mechanosensitive structures. These structures are controlled by small GTPases of the Ras homology (Rho) family. We investigated the effects of RhoA, Rac1, and Cdc42 modulation of osteoblastic cells under microgravity conditions. Human MG-63 osteoblast-like cells silenced for RhoGTPases were cultured in the automated Biobox bioreactor (European Space Agency) aboard the Foton M3 satellite and compared to replicate ground-based controls. The cells were fixed after 69 h of microgravity exposure for postflight analysis of focal contacts, F-actin polymerization, vascular endothelial growth factor (VEGF) expression, and matrix targeting. We found that RhoA silencing did not affect sensitivity to microgravity but that Rac1 and, to a lesser extent, Cdc42 abrogation was particularly efficient in counteracting the spaceflight-related reduction of the number of focal contacts [-50% in silenced, scrambled (SiScr) controls vs. -15% for SiRac1], the number of F-actin fibers (-60% in SiScr controls vs. -10% for SiRac1), and the depletion of matrix-bound VEGF (-40% in SiScr controls vs. -8% for SiRac1). Collectively, these data point out the role of the VEGF/Rho GTPase axis in mechanosensing and validate Rac1-mediated signaling pathways as potential targets for counteracting microgravity effects. PMID:24903274

  16. Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche

    PubMed Central

    Zhang, Wenting; Gu, Yexin; Sun, Qiaoling; Siegel, David S.; Tolias, Peter; Yang, Zheng

    2015-01-01

    We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC) using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1) optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2) replenishing OSB during culture as a practical means of prolonging MMC’s viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses. PMID:25973790

  17. Osteoblasts growth behaviour on bio-based calcium carbonate aragonite nanocrystal.

    PubMed

    Shafiu Kamba, Abdullahi; Zakaria, Zuki Abu Bakar

    2014-01-01

    Calcium carbonate (CaCO3) nanocrystals derived from cockle shells emerge to present a good concert in bone tissue engineering because of their potential to mimic the composition, structure, and properties of native bone. The aim of this study was to evaluate the biological response of CaCO3 nanocrystals on hFOB 1.19 and MC3T3 E-1 osteoblast cells in vitro. Cell viability and proliferation were assessed by MTT and BrdU assays, and LDH was measured to determine the effect of CaCO3 nanocrystals on cell membrane integrity. Cellular morphology was examined by SEM and fluorescence microscopy. The results showed that CaCO3 nanocrystals had no toxic effects to some extent. Cell proliferation, alkaline phosphatase activity, and protein synthesis were enhanced by the nanocrystals when compared to the control. Cellular interactions were improved, as indicated by SEM and fluorescent microscopy. The production of VEGF and TGF-1 was also affected by the CaCO3 nanocrystals. Therefore, bio-based CaCO3 nanocrystals were shown to stimulate osteoblast differentiation and improve the osteointegration process. PMID:24734228

  18. Cellular reactions of osteoblast-like cells to a novel nanocomposite membrane for guided bone regeneration

    NASA Astrophysics Data System (ADS)

    Meng, Yao; Liu, Man; Wang, Shao-An; Mo, An-Chun; Huang, Cui; Zuo, Yi; Li, Ji-Dong

    2008-11-01

    This study investigated the bioactivity and biocompatibility of hydroxyapatite nanoparticles (n-HA)/Polyamide-66 (PA66) nanocomposite membrane and expanded-polytetrafluoroethylene (e-PTFE) membrane (as control) to MG63 osteoblast-like cells. The attachment and proliferation of the cells on the porous surface of nHA/PA66 membrane and the surface of e-PTFE membrane were evaluated by scanning electron microscope (SEM) observation and the MTT assay. The bioactivity of the cells on the surface of the two membranes was evaluated by testing cell viability and alkaline phosphatase (ALP) activities. The results suggested that the bioresponse of MG63 osteoblast-like cells on the porous surface of nHA/PA66 membrane was better than the bioresponse on the opposite surface of e-PTFE membrane. Because of a better cell attachment manner, there is a potential utilization of the guided bone regeneration (GBR) membrane to substitute nHA/PA66 membrane for e-PTFE membrane.

  19. A Comparison of Epithelial Cells, Fibroblasts, and Osteoblasts in Dental Implant Titanium Topographies

    PubMed Central

    Teng, Fu-Yuan; Ko, Chia-Ling; Kuo, Hsien-Nan; Hu, Jin-Jia; Lin, Jia-Horng; Lou, Ching-Wen; Hung, Chun-Cheng; Wang, Yin-Lai; Cheng, Cheng-Yi; Chen, Wen-Cheng

    2012-01-01

    The major challenge for dental implants is achieving optimal esthetic appearance and a concept to fulfill this criterion is evaluated. The key to an esthetically pleasing appearance lies in the properly manage the soft tissue profile around dental implants. A novel implant restoration technique on the surface was proposed as a way to augment both soft- and hard-tissue profiles at potential implant sites. Different levels of roughness can be attained by sandblasting and acid etching, and a tetracalcium phosphate was used to supply the ions. In particular, the early stage attaching and repopulating abilities of bone cell osteoblasts (MC3T3-E1), fibroblasts (NIH 3T3), and epithelial cells (XB-2) were evaluated. The results showed that XB-2 cell adhesive qualities of a smooth surface were better than those of the roughened surfaces, the proliferative properties were reversed. The effects of roughness on the characteristics of 3T3 cells were opposite to the result for XB-2 cells. E1 proliferative ability did not differ with any statistical significance. These results suggest that a rougher surface which provided calcium and phosphate ions have the ability to enhance the proliferation of osteoblast and the inhibition of fibroblast growth that enhance implant success ratios. PMID:22287942

  20. Effects of imatinib and nilotinib on the whole transcriptome of cultured murine osteoblasts

    PubMed Central

    Kirschner, Gyöngyi; Balla, Bernadett; Horváth, Péter; Kövesdi, Andrea; Lakatos, Gergely; Takács, István; Nagy, Zsolt; Tóbiás, Bálint; Árvai, Kristóf; Kósa, János Pál; Lakatos, Péter

    2016-01-01

    Numerous clinical observations have confirmed that breakpoint cluster region-abelson fusion oncoprotein tyrosine kinase inhibitors used in leukemia treatment alter bone physiology in a complex manner. The aim of the present study was to analyze the whole transcriptome of cultured murine osteoblasts and determine the changes following treatment with imatinib and nilotinib using Sequencing by Oligonucleotide Ligation and Detection next generation RNA sequencing. This study also aimed to identify candidate signaling pathways and network regulators by multivariate Ingenuity Pathway Analysis. Based on the right-tailed Fisher's exact test, significantly altered pathways including upstream regulators were defined for each drug. The correlation between these pathways and bone metabolism was also examined. The preliminary results suggest the two drugs have different mechanisms of action on osteoblasts, and imatinib was shown to have a greater effect on gene expression. Data also indicated the potential role of a number of genes and signaling cascades that may contribute to identifying novel targets for the treatment of metabolic bone diseases. PMID:27430367

  1. Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells.

    PubMed

    Guignandon, Alain; Faure, Céline; Neutelings, Thibaut; Rattner, Aline; Mineur, Pierre; Linossier, Marie-Thérèse; Laroche, Norbert; Lambert, Charles; Deroanne, Christophe; Nusgens, Betty; Demets, René; Colige, Alain; Vico, Laurence

    2014-09-01

    Bone cells exposed to real microgravity display alterations of their cytoskeleton and focal adhesions, two major mechanosensitive structures. These structures are controlled by small GTPases of the Ras homology (Rho) family. We investigated the effects of RhoA, Rac1, and Cdc42 modulation of osteoblastic cells under microgravity conditions. Human MG-63 osteoblast-like cells silenced for RhoGTPases were cultured in the automated Biobox bioreactor (European Space Agency) aboard the Foton M3 satellite and compared to replicate ground-based controls. The cells were fixed after 69 h of microgravity exposure for postflight analysis of focal contacts, F-actin polymerization, vascular endothelial growth factor (VEGF) expression, and matrix targeting. We found that RhoA silencing did not affect sensitivity to microgravity but that Rac1 and, to a lesser extent, Cdc42 abrogation was particularly efficient in counteracting the spaceflight-related reduction of the number of focal contacts [-50% in silenced, scrambled (SiScr) controls vs. -15% for SiRac1], the number of F-actin fibers (-60% in SiScr controls vs. -10% for SiRac1), and the depletion of matrix-bound VEGF (-40% in SiScr controls vs. -8% for SiRac1). Collectively, these data point out the role of the VEGF/Rho GTPase axis in mechanosensing and validate Rac1-mediated signaling pathways as potential targets for counteracting microgravity effects.

  2. Carbon nanohorns allow acceleration of osteoblast differentiation via macrophage activation

    NASA Astrophysics Data System (ADS)

    Hirata, Eri; Miyako, Eijiro; Hanagata, Nobutaka; Ushijima, Natsumi; Sakaguchi, Norihito; Russier, Julie; Yudasaka, Masako; Iijima, Sumio; Bianco, Alberto; Yokoyama, Atsuro

    2016-07-01

    Carbon nanohorns (CNHs), formed by a rolled graphene structure and terminating in a cone, are promising nanomaterials for the development of a variety of biological applications. Here we demonstrate that alkaline phosphatase activity is dramatically increased by coculture of human monocyte derived macrophages (hMDMs) and human mesenchymal stem cells (hMSCs) in the presence of CNHs. CNHs were mainly localized in the lysosome of macrophages more than in hMSCs during coculturing. At the same time, the amount of Oncostatin M (OSM) in the supernatant was also increased during incubation with CNHs. Oncostatin M (OSM) from activated macrophage has been reported to induce osteoblast differentiation and matrix mineralization through STAT3. These results suggest that the macrophages engulfed CNHs and accelerated the differentiation of mesenchymal stem cells into the osteoblast via OSM release. We expect that the proof-of-concept on the osteoblast differentiation capacity by CNHs will allow future studies focused on CNHs as ideal therapeutic materials for bone regeneration.Carbon nanohorns (CNHs), formed by a rolled graphene structure and terminating in a cone, are promising nanomaterials for the development of a variety of biological applications. Here we demonstrate that alkaline phosphatase activity is dramatically increased by coculture of human monocyte derived macrophages (hMDMs) and human mesenchymal stem cells (hMSCs) in the presence of CNHs. CNHs were mainly localized in the lysosome of macrophages more than in hMSCs during coculturing. At the same time, the amount of Oncostatin M (OSM) in the supernatant was also increased during incubation with CNHs. Oncostatin M (OSM) from activated macrophage has been reported to induce osteoblast differentiation and matrix mineralization through STAT3. These results suggest that the macrophages engulfed CNHs and accelerated the differentiation of mesenchymal stem cells into the osteoblast via OSM release. We expect that the

  3. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

    SciTech Connect

    Kawano, Michinao; Ariyoshi, Wataru; Iwanaga, Kenjiro; Okinaga, Toshinori; Habu, Manabu; Yoshioka, Izumi; Tominaga, Kazuhiro; Nishihara, Tatsuji

    2011-02-25

    Research highlights: {yields} In this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. {yields} MG63 cells were incubated with BMP-2 and HA for various time periods. {yields} Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. {yields} HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation. -- Abstract: Objectives: Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. Materials and methods: Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR. Results: BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and

  4. Cardiac differentiation potential of human induced pluripotent stem cells in a 3D self-assembling peptide scaffold.

    PubMed

    Puig-Sanvicens, Veronica A C; Semino, Carlos E; Zur Nieden, Nicole I

    2015-01-01

    In the past decade, various strategies for cardiac reparative medicine involving stem cells from multiple sources have been investigated. However, the intra-cardiac implantation of cells with contractile ability may seriously disrupt the cardiac syncytium and de-synchronize cardiac rhythm. For this reason, bioactive cardiac implants, consisting of stem cells embedded in biomaterials that act like band aids, have been exploited to repair the cardiac wall after myocardial infarction. For such bioactive implants to function properly after transplantation, the choice of biomaterial is equally important as the selection of the stem cell source. While adult stem cells have shown promising results, they have various disadvantages including low proliferative potential in vitro, which make their successful usage in human transplants difficult. As a first step towards the development of a bioactive cardiac patch, we investigate here the cardiac differentiation properties of human induced pluripotent stem cells (hiPSCs) when cultured with and without ascorbic acid (AA) and when embedded in RAD16-I, a biomaterial commonly used to develop cardiac implants. In adherent cultures and in the absence of RAD16-I, AA promotes the cardiac differentiation of hiPSCs by enhancing the expression of specific cardiac genes and proteins and by increasing the number of contracting clusters. In turn, embedding in peptide hydrogel based on RAD16-I interferes with the normal cardiac differentiation progression. Embedded hiPSCs up-regulate genes associated with early cardiogenesis by up to 105 times independently of the presence of AA. However, neither connexin 43 nor troponin I proteins, which are related with mature cardiomyocytes, were detected and no contraction was noted in the constructs. Future experiments will need to focus on characterizing the mature cardiac phenotype of these cells when implanted into infarcted myocardia and assess their regenerative potential in vivo. PMID:26707885

  5. Stem cell factor (SCF) protects osteoblasts from oxidative stress through activating c-Kit-Akt signaling

    SciTech Connect

    Yang, Lei; Wu, Zhong; Yin, Gang; Liu, Haifeng; Guan, Xiaojun; Zhao, Xiaoqiang; Wang, Jianguang; Zhu, Jianguo

    2014-12-12

    Highlights: • SCF receptor c-Kit is functionally expressed in primary and transformed osteoblasts. • SCF protects primary and transformed osteoblasts from H{sub 2}O{sub 2}. • SCF activation of c-Kit in osteoblasts, required for its cyto-protective effects. • c-Kit mediates SCF-induced Akt activation in cultured osteoblasts. • Akt activation is required for SCF-regulated cyto-protective effects in osteoblasts. - Abstract: Osteoblasts regulate bone formation and remodeling, and are main target cells of oxidative stress in the progression of osteonecrosis. The stem cell factor (SCF)-c-Kit pathway plays important roles in the proliferation, differentiation and survival in a range of cell types, but little is known about its functions in osteoblasts. In this study, we found that c-Kit is functionally expressed in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Its ligand SCF exerted significant cyto-protective effects against hydrogen peroxide (H{sub 2}O{sub 2}). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H{sub 2}O{sub 2}. Pharmacological inhibition (by Imatinib and Dasatinib) or shRNA-mediated knockdown of c-Kit thus inhibited SCF-mediated osteoblast protection. Further investigations showed that protection by SCF against H{sub 2}O{sub 2} was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H{sub 2}O{sub 2} activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H{sub 2}O{sub 2}-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.

  6. Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

    PubMed

    Wu, Li-An; Wang, Feng; Donly, Kevin J; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary; Chen, Shuo

    2016-06-01

    Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. PMID:26595646

  7. Cancer–Osteoblast Interaction Reduces Sost Expression in Osteoblasts and Up-Regulates lncRNA MALAT1 in Prostate Cancer

    PubMed Central

    Sebastian, Aimy; Hum, Nicholas R.; Hudson, Bryan D.; Loots, Gabriela G.

    2015-01-01

    Dynamic interaction between prostate cancer and the bone microenvironment is a major contributor to metastasis of prostate cancer to bone. In this study, we utilized an in vitro co-culture model of PC3 prostate cancer cells and osteoblasts followed by microarray based gene expression profiling to identify previously unrecognized prostate cancer–bone microenvironment interactions. Factors secreted by PC3 cells resulted in the up-regulation of many genes in osteoblasts associated with bone metabolism and cancer metastasis, including Mmp13, Il-6 and Tgfb2, and down-regulation of Wnt inhibitor Sost. To determine whether altered Sost expression in the bone microenvironment has an effect on prostate cancer metastasis, we co-cultured PC3 cells with Sost knockout (SostKO) osteoblasts and wildtype (WT) osteoblasts and identified several genes differentially regulated between PC3-SostKO osteoblast co-cultures and PC3-WT osteoblast co-cultures. Co-culturing PC3 cells with WT osteoblasts up-regulated cancer-associated long noncoding RNA (lncRNA) MALAT1 in PC3 cells. MALAT1 expression was further enhanced when PC3 cells were co-cultured with SostKO osteoblasts and treatment with recombinant Sost down-regulated MALAT1 expression in these cells. Our results suggest that reduced Sost expression in the tumor microenvironment may promote bone metastasis by up-regulating MALAT1 in prostate cancer. PMID:27600237

  8. Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

    PubMed

    Wu, Li-An; Wang, Feng; Donly, Kevin J; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary; Chen, Shuo

    2016-06-01

    Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages.

  9. Establishment of Immortalized BMP2/4 Double Knock‐Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis

    PubMed Central

    Wu, Li‐An; Wang, Feng; Donly, Kevin J.; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary

    2015-01-01

    Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock‐out (ko/ko) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4ko/ko cells were verified by green immunofluorescence and PCR. BMP2/4ko/ko osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone‐relate genes was reduced in the BMP2/4ko/ko cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4ko/ko osteoblasts as reflected by decreased Mmp‐2 and Mmp‐9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. J. Cell. Physiol. 231: 1189–1198, 2016. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:26595646

  10. Cancer–Osteoblast Interaction Reduces Sost Expression in Osteoblasts and Up-Regulates lncRNA MALAT1 in Prostate Cancer

    PubMed Central

    Sebastian, Aimy; Hum, Nicholas R.; Hudson, Bryan D.; Loots, Gabriela G.

    2015-01-01

    Dynamic interaction between prostate cancer and the bone microenvironment is a major contributor to metastasis of prostate cancer to bone. In this study, we utilized an in vitro co-culture model of PC3 prostate cancer cells and osteoblasts followed by microarray based gene expression profiling to identify previously unrecognized prostate cancer–bone microenvironment interactions. Factors secreted by PC3 cells resulted in the up-regulation of many genes in osteoblasts associated with bone metabolism and cancer metastasis, including Mmp13, Il-6 and Tgfb2, and down-regulation of Wnt inhibitor Sost. To determine whether altered Sost expression in the bone microenvironment has an effect on prostate cancer metastasis, we co-cultured PC3 cells with Sost knockout (SostKO) osteoblasts and wildtype (WT) osteoblasts and identified several genes differentially regulated between PC3-SostKO osteoblast co-cultures and PC3-WT osteoblast co-cultures. Co-culturing PC3 cells with WT osteoblasts up-regulated cancer-associated long noncoding RNA (lncRNA) MALAT1 in PC3 cells. MALAT1 expression was further enhanced when PC3 cells were co-cultured with SostKO osteoblasts and treatment with recombinant Sost down-regulated MALAT1 expression in these cells. Our results suggest that reduced Sost expression in the tumor microenvironment may promote bone metastasis by up-regulating MALAT1 in prostate cancer.

  11. Strontium ranelate improves the interaction of osteoblastic cells with titanium substrates: Increase in cell proliferation, differentiation and matrix mineralization.

    PubMed

    Querido, William; Farina, Marcos; Anselme, Karine

    2015-01-01

    We describe direct effects of strontium ranelate on the interaction of osteoblastic cells with different titanium substrates. Our goal was to better understand the potential of this drug for improving the efficacy of bone implants. Treatment was done with 0.12 and 0.5 mM Sr(2+) of strontium ranelate in cell culture. We analyzed cell response to the drug on titanium substrates with surface topographies obtained using acid etching, electro-erosion processing, sandblasting, and machine-tooling. Treatment preserved the initial cell adhesion to the substrates, cell shape parameters (area, aspect ratio, circularity, and solidity), and the orientation of cells on grooved surfaces. However, both concentrations of the drug increased cell proliferation in all substrates. Moreover, a dose-dependent increase in alkaline phosphatase activity and in the production of mineralized matrix with typical features of bone tissue was shown. The observed effects were similar in the different substrates. In conclusion, strontium ranelate improved the interaction of osteoblastic cells with titanium substrates, increasing cell proliferation and differentiation into mature osteoblasts and the production of bone-like mineralized matrix for all substrates. This study highlights a promising role of strontium ranelate on enhancing the clinical success of bone implants, particularly in patients with osteoporosis. PMID:26176488

  12. Strontium ranelate improves the interaction of osteoblastic cells with titanium substrates: Increase in cell proliferation, differentiation and matrix mineralization

    PubMed Central

    Querido, William; Farina, Marcos; Anselme, Karine

    2015-01-01

    We describe direct effects of strontium ranelate on the interaction of osteoblastic cells with different titanium substrates. Our goal was to better understand the potential of this drug for improving the efficacy of bone implants. Treatment was done with 0.12 and 0.5 mM Sr2+ of strontium ranelate in cell culture. We analyzed cell response to the drug on titanium substrates with surface topographies obtained using acid etching, electro-erosion processing, sandblasting, and machine-tooling. Treatment preserved the initial cell adhesion to the substrates, cell shape parameters (area, aspect ratio, circularity, and solidity), and the orientation of cells on grooved surfaces. However, both concentrations of the drug increased cell proliferation in all substrates. Moreover, a dose-dependent increase in alkaline phosphatase activity and in the production of mineralized matrix with typical features of bone tissue was shown. The observed effects were similar in the different substrates. In conclusion, strontium ranelate improved the interaction of osteoblastic cells with titanium substrates, increasing cell proliferation and differentiation into mature osteoblasts and the production of bone-like mineralized matrix for all substrates. This study highlights a promising role of strontium ranelate on enhancing the clinical success of bone implants, particularly in patients with osteoporosis. PMID:26176488

  13. Strontium ranelate improves the interaction of osteoblastic cells with titanium substrates: Increase in cell proliferation, differentiation and matrix mineralization.

    PubMed

    Querido, William; Farina, Marcos; Anselme, Karine

    2015-01-01

    We describe direct effects of strontium ranelate on the interaction of osteoblastic cells with different titanium substrates. Our goal was to better understand the potential of this drug for improving the efficacy of bone implants. Treatment was done with 0.12 and 0.5 mM Sr(2+) of strontium ranelate in cell culture. We analyzed cell response to the drug on titanium substrates with surface topographies obtained using acid etching, electro-erosion processing, sandblasting, and machine-tooling. Treatment preserved the initial cell adhesion to the substrates, cell shape parameters (area, aspect ratio, circularity, and solidity), and the orientation of cells on grooved surfaces. However, both concentrations of the drug increased cell proliferation in all substrates. Moreover, a dose-dependent increase in alkaline phosphatase activity and in the production of mineralized matrix with typical features of bone tissue was shown. The observed effects were similar in the different substrates. In conclusion, strontium ranelate improved the interaction of osteoblastic cells with titanium substrates, increasing cell proliferation and differentiation into mature osteoblasts and the production of bone-like mineralized matrix for all substrates. This study highlights a promising role of strontium ranelate on enhancing the clinical success of bone implants, particularly in patients with osteoporosis.

  14. Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment.

    PubMed

    Reichert, Johannes C; Quent, Verena M C; Burke, Leslie J; Stansfield, Scott H; Clements, Judith A; Hutmacher, Dietmar W

    2010-11-01

    Prostate cancer metastasis is reliant on the reciprocal interactions between cancer cells and the bone niche/micro-environment. The production of suitable matrices to study metastasis, carcinogenesis and in particular prostate cancer/bone micro-environment interaction has been limited to specific protein matrices or matrix secreted by immortalised cell lines that may have undergone transformation processes altering signaling pathways and modifying gene or receptor expression. We hypothesize that matrices produced by primary human osteoblasts are a suitable means to develop an in vitro model system for bone metastasis research mimicking in vivo conditions. We have used a decellularized matrix secreted from primary human osteoblasts as a model for prostate cancer function in the bone micro-environment. We show that this collagen I rich matrix is of fibrillar appearance, highly mineralized, and contains proteins, such as osteocalcin, osteonectin and osteopontin, and growth factors characteristic of bone extracellular matrix (ECM). LNCaP and PC3 cells grown on this matrix, adhere strongly, proliferate, and express markers consistent with a loss of epithelial phenotype. Moreover, growth of these cells on the matrix is accompanied by the induction of genes associated with attachment, migration, increased invasive potential, Ca(2+) signaling and osteolysis. In summary, we show that growth of prostate cancer cells on matrices produced by primary human osteoblasts mimics key features of prostate cancer bone metastases and thus is a suitable model system to study the tumor/bone micro-environment interaction in this disease.

  15. A combination of CO2 laser and plasma surface modification of poly(etheretherketone) to enhance osteoblast response

    NASA Astrophysics Data System (ADS)

    Zheng, Yanyan; Xiong, Chengdong; Wang, Zhecun; Li, Xiaoyu; Zhang, Lifang

    2015-07-01

    Poly(etheretherketone) (PEEK) is a rigid semicrystalline polymer that combines excellent mechanical properties, broad chemical resistance and bone-like stiffness and is widely used in biomedical fields. However, the bio-inert surface of PEEK tends to hinder its biomedical applications when direct osteointegration between the implants and the host tissue is desired. In this work, we demonstrate a dual modification method, which combines the laser and plasma surface treatment to combine advantages of both chemical states and microstructures for osteoblasts responses. While the plasma treatment introduces surface carboxyl groups (sbnd COOH) onto PEEK surface, the laser treatment constructs microstructures over the PEEK surface. Our results indicated that sbnd COOH as well as microgrooves containing micropores or microcraters structure are constructed on PEEK surface and plasma treatment has no apparent effect on the morphology of microstructures produced by laser micromachining. Unexpectedly, the superior mechanical properties of PEEK were maintained irrespective of the treatment used. Compared to native PEEK and single treated PEEK, dual modified PEEK is more favorable for pre-osteoblasts (MC3T3-E1) adhesion, spreading and proliferation. Moreover, cell pseudopodia protrude into the micropores or microcraters, in favor of forming firmer bone-implant integration. Our study illustrates enhanced osteoblasts responses to dual treated PEEK surface, which gives beneficial information of its potential use in orthopedic or dental implants.

  16. hBMP-7 induces the differentiation of adipose-derived mesenchymal stem cells into osteoblast-like cells.

    PubMed

    Ren, Y; Han, C; Wang, J; Jia, Y; Kong, L; Eerdun, T; Wu, L; Jiang, D

    2016-01-01

    The aim of this study was to investigate the differentiation potential of adipose-derived mesenchymal stem cells (ADMSCs) into osteoblasts by human bone morphogenetic protein-7 (hBMP-7) induction. ADMSCs were isolated from the subcutaneous adipose tissue of a rabbit, and then transfected with the pcDNA3.1 vector alone and pcDNA3.1-hBMP-7 (hBMP-7), respectively. Untransfected ADMSCs were used as the control group. After transfection, the morphology and green fluorescent protein (GFP) fluorescence intensity of ADMSCs were observed by fluorescent microscopy. The 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay was performed to detect the growth of ADMSCs at 1, 3, and 5 days, respectively. Transmission electron microscopy was performed to observe the ultrastructural morphology of ADMSCs. In addition, ADMSCs were stained with quinalizarin and toluidine blue to reflect the content of osteoblasts and chondrocytes, respectively. Finally, the expression of collagen I and osteocalcin in ADMSCs was detected by western blot. ADMSCs were successfully isolated. Obvious GFP fluorescence and high expression of hBMP-7 demonstrated the successful transfection of hBMP-7. Specific morphological characters with a metabolically active ultrastructure were exhibited on the ADMSCs transfected with hBMP- 7. In addition, the growth rate of ADMSCs transfected with hBMP-7 was significantly higher than that of the cells in the vector and control groups. Successfully induced osteoblast-like cells were identified by an obvious erythrine area and high expression of collagen I and osteocalcin in ADMSCs transfected with hBMP-7. Thus, ADMSCs can be successfully differentiated into osteoblast-like cells by hBMP-7 induction in vitro. PMID:27525862

  17. Effect of Q-switched Laser Surface Texturing of Titanium on Osteoblast Cell Response

    NASA Astrophysics Data System (ADS)

    Voisey, K. T.; Scotchford, C. A.; Martin, L.; Gill, H. S.

    Titanium and its alloys are important biomedical materials. It is known that the surface texture of implanted medical devices affects cell response. Control of cell response has the potential to enhance fixation of implants into bone and, in other applications, to prevent undesired cell adhesion. The potential use of a 100W Q-switched YAG laser miller (DMG Lasertec 60 HSC) for texturing titanium is investigated. A series of regular features with dimensions of the order of tens of micrometers are generated in the surface of titanium samples and the cell response to these features is determined. Characterisation of the laser milled features reveals features with a lengthscale of a few microns superposed on the larger scale structures, this is attributed to resolidification of molten droplets generated and propelled over the surface by individual laser pulses. The laser textured samples are exposed to osteoblast cells and it is seen that cells do respond to the features in the laser textured surfaces.

  18. Effects of Load on Normal Human Osteoblast Function

    NASA Astrophysics Data System (ADS)

    Reseland, J. E.; Devakottai, Sundar; Sundaresan, A.

    2013-02-01

    The effects of load on the secretion and expression of bone markers were tested at different stages of differentiation of primary human osteoblasts. NHOs were both seeded with and without cytodex 3 beads (Sigma),transferred to a NASA rotating wall vessel (modeled microgravity) and harvested at day 7 and day 14. Differentiated and undifferentiated NHOs were loaded at 6-50G for 30 min and compared to cells incubated at 1G after 1 day and 3 days. Collectively the results demonstrate that load has a differential effect on osteoblast differentiation as seen in modeled microgravity and shows specificity in expression of bone cell markers vs. expression of secreted paracrine signaling markers.

  19. Rebamipide Delivered by Brushite Cement Enhances Osteoblast and Macrophage Proliferation

    PubMed Central

    Pujari-Palmer, Michael; Pujari-Palmer, Shiuli; Engqvist, Håkan; Karlsson Ott, Marjam

    2015-01-01

    Many of the bioactive agents capable of stimulating osseous regeneration, such as bone morphogenetic protein-2 (BMP-2) or prostaglandin E2 (PGE2), are limited by rapid degradation, a short bioactive half-life at the target site in vivo, or are prohibitively expensive to obtain in large quantities. Rebamipide, an amino acid modified hydroxylquinoline, can alter the expression of key mediators of bone anabolism, cyclo-oxygenase 2 (COX-2), BMP-2 and vascular endothelial growth factor (VEGF), in diverse cell types such as mucosal and endothelial cells or chondrocytes. The present study investigates whether Rebamipide enhances proliferation and differentiation of osteoblasts when delivered from brushite cement. The reactive oxygen species (ROS) quenching ability of Rebampide was tested in macrophages as a measure of bioactivity following drug release incubation times, up to 14 days. Rebamipide release from brushite occurrs via non-fickian diffusion, with a rapid linear release of 9.70% ±0.37% of drug per day for the first 5 days, and an average of 0.5%-1% per day thereafter for 30 days. Rebamipide slows the initial and final cement setting time by up to 3 and 1 minute, respectively, but does not significantly reduce the mechanical strength below 4% (weight percentage). Pre-osteoblast proliferation increases by 24% upon exposure to 0.4uM Rebamipide, and by up to 73% when Rebamipide is delivered via brushite cement. Low doses of Rebamipide do not adversely affect peak alkaline phosphatase activity in differentiating pre-osteoblasts. Rebamipide weakly stimulates proliferation in macrophages at low concentrations (118 ±7.4% at 1uM), and quenches ROS by 40-60%. This is the first investigation of Rebamipide in osteoblasts. PMID:26023912

  20. PTH and PTHrP Signaling in Osteoblasts

    PubMed Central

    Datta, Nabanita S.; Abou-Samra, Abdul B.<