scholarly journals Connexin 43 Affects Osteogenic Differentiation of the Posterior Longitudinal Ligament Cells via Regulation of ERK Activity by Stabilizing Runx2 in Ossification

2016 ◽  
Vol 38 (1) ◽  
pp. 237-247 ◽  
Author(s):  
Haisong Yang ◽  
Lei Shi ◽  
Guodong Shi ◽  
Yongfei Guo ◽  
Dechun Chen ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Connexin 43 ◽  
Posterior Longitudinal Ligament ◽  
Luciferase Reporter ◽  
Dexamethasone Treatment ◽  
Extracellular Signal ◽  
Related Transcription Factor ◽  
Erk Activity ◽  
Junction Proteins

Aims: Connexin 43 is one of the most potent gap junction proteins related to osteoblast differentiation and bone formation. We hypothesized that Connexin 43 is a significant factor in osteogenic differentiation in the posterior longitudinal ligament through the regulation of extracellular signal-regulated kinases (ERK) activity by converging on Runt-related transcription factor 2 (Runx2) activity. In this study, we mapped the activity of Connexin 43 to ERK and Runx2 by extracting longitudinal ligament cell for culture and silencing Connexin expression in addition to dexamethasone treatment in vitro. Methods: qRT-PCR, Western Blot, and Runx2-responsive Luciferase Reporter Assay were performed to detect the activity of ERK, Runx2 and the expression levels of osseous genes under Connexin 43 modification. Results: Downregulation of Connexin 43 resulted in suppression of dexamethasone-induced osteogenic differentiation, inhibition of the ERK and Runx2 activity, and reduction of osseous gene expression. Conclusion: these data support that Connexin 43 significantly regulates osteogenic differentiation in the cells from posterior longitudinal ligament by altering the activity of ERK, and subsequently causing the modification of Runx2.

scholarly journals Osteogenic Properties of 3D-Printed Silica-Carbon-Calcite Composite Scaffolds: Novel Approach for Personalized Bone Tissue Regeneration

2021 ◽  
Vol 22 (2) ◽  
pp. 475
Author(s):  
Parastoo Memarian ◽  
Francesco Sartor ◽  
Enrico Bernardo ◽  
Hamada Elsayed ◽  
Batur Ercan ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Bone Tissue ◽  
Cell Attachment ◽  
Three Dimensional ◽  
Bone Tissue Regeneration ◽  
Hemolysis Assay ◽  
Novel Approach ◽  
Related Transcription Factor ◽  
Diphenyl Tetrazolium Bromide

Carbon enriched bioceramic (C-Bio) scaffolds have recently shown exceptional results in terms of their biological and mechanical properties. The present study aims at assessing the ability of the C-Bio scaffolds to affect the commitment of canine adipose-derived mesenchymal stem cells (cAD-MSCs) and investigating the influence of carbon on cell proliferation and osteogenic differentiation of cAD-MSCs in vitro. The commitment of cAD-MSCs to an osteoblastic phenotype has been evaluated by expression of several osteogenic markers using real-time PCR. Biocompatibility analyses through 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH) activity, hemolysis assay, and Ames test demonstrated excellent biocompatibility of both materials. A significant increase in the extracellular alkaline phosphatase (ALP) activity and expression of runt-related transcription factor (RUNX), ALP, osterix (OSX), and receptor activator of nuclear factor kappa-Β ligand (RANKL) genes was observed in C-Bio scaffolds compared to those without carbon (Bio). Scanning electron microscopy (SEM) demonstrated excellent cell attachment on both material surfaces; however, the cellular layer on C-Bio fibers exhibited an apparent secretome activity. Based on our findings, graphene can improve cell adhesion, growth, and osteogenic differentiation of cAD-MSCs in vitro. This study proposed carbon as an additive for a novel three-dimensional (3D)-printable biocompatible scaffold which could become the key structural material for bone tissue reconstruction.

scholarly journals IQGAP1 Is a Scaffold for Mitogen-Activated Protein Kinase Signaling

2005 ◽  
Vol 25 (18) ◽  
pp. 7940-7952 ◽  
Author(s):  
Monideepa Roy ◽  
Zhigang Li ◽  
David B. Sacks
Keyword(s):  
Growth Factor ◽  
Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Signaling ◽  
Cellular Functions ◽  
Molecular Scaffold ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Erk Activity

ABSTRACT IQGAP1 modulates many cellular functions such as cell-cell adhesion, transcription, cytoskeletal architecture, and selected signaling pathways. We previously documented that IQGAP1 binds extracellular signal-regulated kinase (ERK) 2 and regulates growth factor-stimulated ERK activity. Here we show that MEK, the molecule immediately upstream of ERK in the Ras/mitogen-activated protein (MAP) kinase signaling cascade, also interacts directly with IQGAP1. Both MEK1 and MEK2 bound IQGAP1 in vitro and coimmunoprecipitated with IQGAP1. The addition of ERK2 enhanced by fourfold the in vitro interaction of MEK2 with IQGAP1 without altering binding of MEK1. Similarly, ERK1 promoted MEK binding to IQGAP1, but either MEK protein altered the association between IQGAP1 and ERK. Epidermal growth factor (EGF) differentially regulated binding, enhancing MEK1 interaction while reducing MEK2 binding to IQGAP1. In addition, both knockdown and overexpression of IQGAP1 reduced EGF-stimulated activation of MEK and ERK. Analyses with selective IQGAP1 mutant constructs indicated that MEK binding is crucial for IQGAP1 to modulate EGF activation of ERK. Our data strongly suggest that IQGAP1 functions as a molecular scaffold in the Ras/MAP kinase pathway.

scholarly journals Photoperiod-Dependent Effects of 4-tert-Octylphenol on Adherens and Gap Junction Proteins in Bank Vole Seminiferous Tubules

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Anna Hejmej ◽  
Malgorzata Kotula-Balak ◽  
Katarzyna Chojnacka ◽  
Paulina Kuras ◽  
Marta Lydka-Zarzycka ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Gap Junction ◽  
Bank Vole ◽  
Connexin 43 ◽  
Biological Effects ◽  
Seminiferous Tubules ◽  
Long Term Treatment ◽  
Gap Junction Proteins ◽  
Junction Proteins

In the present study we evaluatedin vivoandin vitroeffects of 4-tert-octylphenol (OP) on the expression and distribution of adherens and gap junction proteins, N-cadherin,β-catenin, and connexin 43 (Cx43), in testes of seasonally breeding rodents, bank voles. We found that in bank vole testes expression and distribution of N-cadherin,β-catenin, and Cx43 were photoperiod dependent. Long-term treatment with OP (200 mg/kg b.w.) resulted in the reduction of junction proteins expressions (P<0.05,P<0.01) and their delocalization in the testes of males kept in long photoperiod, whereas in short-day animals slight increase of Cx43 (P<0.05), N-cadherin, andβ-catenin (statistically nonsignificant) levels was observed. Effects of OP appeared to be independent of FSH and were maintained duringin vitroorgan culture, indicating that OP acts directly on adherens and gap junction proteins in the testes. An experiment performed using an antiestrogen ICI 182,780 demonstrated that the biological effects of OP onβ-catenin and Cx43 involve an estrogen receptor-mediated response. Taken together, in bank vole organization of adherens and gap junctions and their susceptibility to OP are related to the length of photoperiod. Alterations in cadherin/catenin and Cx43-based junction may partially result from activation of estrogen receptorαand/orβsignaling pathway.

scholarly journals SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Antagonizing DNMT1

2021 ◽  
Vol 9 ◽  
Author(s):  
Bo Jia ◽  
Jun Chen ◽  
Qin Wang ◽  
Xiang Sun ◽  
Jiusong Han ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Notch Signaling ◽  
Osteogenic Differentiation ◽  
Dna Methyltransferases ◽  
Luciferase Reporter ◽  
Calcium Deposition ◽  
Alizarin Red

BackgroundAdipose-derived stem cells (ADSCs) are increasingly used in regenerative medicine because of their potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin protein 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that plays important roles in cell differentiation. NOTCH signaling has also been reported to involve in osteogenic differentiation. However, the function of SIRT6 in osteogenic differentiation of ADSCs and its relation to the NOTCH signaling pathways are yet to be explored.MethodsThe in vitro study with human ADSCs (hADSCs) and in vivo experiments with nude mice have been performed. Alkaline phosphatase (ALP) assays and ALP staining were used to detect osteogenic activity. Alizarin Red staining was performed to detect calcium deposition induced by osteogenic differentiation of ADSCs. Western blot, RT-qPCR, luciferase reporter assay, and co-immunoprecipitation assay were applied to explore the relationship between of SIRT6, DNA methyltransferases (DNMTs) and NOTCHs.ResultsSIRT6 promoted ALP activity, enhanced mineralization and upregulated expression of osteogenic-related genes of hADSCs in vitro and in vivo. Further mechanistic studies showed that SIRT6 deacetylated DNMT1, leading to its unstability at protein level. The decreased expression of DNMT1 prevented the abnormal DNA methylation of NOTCH1 and NOTCH2, resulting in the upregulation of their transcription. SIRT6 overexpression partially suppressed the abnormal DNA methylation of NOTCH1 and NOTCH2 by antagonizing DNMT1, leading to an increased capacity of ADSCs for their osteogenic differentiation.ConclusionThis study demonstrates that SIRT6 physical interacts with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein, leading to the activation of NOTCH1 and NOTCH2, Which in turn promotes the osteogenic differentiation of ADSCs.

scholarly journals Let-7c regulates proliferation and osteodifferentiation of human adipose-derived mesenchymal stem cells under oxidative stress by targeting SCD-1

2019 ◽  
Vol 316 (1) ◽  
pp. C57-C69 ◽  
Author(s):  
Zihui Zhou ◽  
Yuanshan Lu ◽  
Yao Wang ◽  
Lin Du ◽  
Yunpeng Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Postmenopausal Osteoporosis ◽  
Luciferase Reporter ◽  
Matrix Mineralization ◽  
Protein Levels ◽  
Micro Rnas

Osteoporosis is a progressive bone disease characterized by decreased bone mass and density, which usually parallels a reduced antioxidative capacity and increased reactive oxygen species formation. Adipose-derived mesenchymal stem cells (ADMSCs), a population of self-renewing multipotent cells, are a well-recognized source of potential bone precursors with significant clinical potential for tissue regeneration. We previously showed that overexpressing stearoyl-CoA desaturase 1 (SCD-1) promotes osteogenic differentiation of mesenchymal stem cells. Micro-RNAs (miRNAs) are noncoding RNAs recently recognized to play key roles in many developmental processes, and miRNA let-7c is downregulated during osteoinduction. We found that let-7c was upregulated in the serum of patients with postmenopausal osteoporosis compared with healthy controls. Levels of let-7c during osteogenic differentiation of ADMSCs were examined under oxidative stress in vitro and found to be upregulated. Overexpression of let-7c inhibited osteogenic differentiation, whereas inhibition of let-7c function promoted this process, evidenced by increased expression of osteoblast-specific genes, alkaline phosphatase activity, and matrix mineralization. The luciferase reporter assay was used to validate SCD-1 as a target of let-7c. Further experiments showed that silencing of SCD-1 significantly attenuated the effect of let-7c inhibitor on osteoblast markers, providing strong evidence that let-7c modulates osteogenic differentiation by targeting SCD-1. Inhibition of let-7c promoted the translocation of β-catenin into nuclei, thus activating Wnt/β-catenin signaling. Collectively, these data suggest that let-7c is induced under oxidative stress conditions and in osteoporosis, reducing SCD-1 protein levels, switching off Wnt/β-catenin signaling, and inhibiting osteogenic differentiation. Thus, let-7c may be a potential therapeutic target in the treatment of osteoporosis and especially postmenopausal osteoporosis.

scholarly journals 12-O-Tetradecanoylphorbol-13-acetate increases cardiomyogenesis through PKC/ERK signaling

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Katarzyna Anna Radaszkiewicz ◽  
Deborah Beckerová ◽  
Lucie Woloszczuková ◽  
Tomasz Witold Radaszkiewicz ◽  
Petra Lesáková ◽  
...  
Keyword(s):  
Cellular Response ◽  
Embryonic Stem ◽  
Kinase C ◽  
Differentiation Protocol ◽  
Extracellular Signal ◽  
Cell Differentiation Factor ◽  
Canonical Wnt ◽  
Erk Activity ◽  
Mes Cells

Abstract 12-O-Tetradecanoylphorbol-13-acetate (TPA) is the most widely used diacylglycerol (DAG) mimetic agent and inducer of protein kinase C (PKC)-mediated cellular response in biomedical studies. TPA has been proposed as a pluripotent cell differentiation factor, but results obtained have been inconsistent. In the present study we show that TPA can be applied as a cardiomyogenesis-promoting factor for the differentiation of mouse embryonic stem (mES) cells in vitro. The mechanism of TPA action is mediated by the induction of extracellular signal-regulated kinase (ERK) activity and the subsequent phosphorylation of GATA4 transcription factor. Interestingly, general mitogens (FGF, EGF, VEGF and serum) or canonical WNT signalling did not mimic the effect of TPA. Moreover, on the basis of our results, we postulate that a TPA-sensitive population of cardiac progenitor cells exists at a certain time point (after days 6–8 of the differentiation protocol) and that the proposed treatment can be used to increase the multiplication of ES cell-derived cardiomyocytes.

scholarly journals PDCD10 Interacts with Ste20-related Kinase MST4 to Promote Cell Growth and Transformation via Modulation of the ERK Pathway

2007 ◽  
Vol 18 (6) ◽  
pp. 1965-1978 ◽  
Author(s):  
Xi Ma ◽  
Hongshan Zhao ◽  
Jingxuan Shan ◽  
Feng Long ◽  
Yaoyao Chen ◽  
...  
Keyword(s):  
Cell Growth ◽  
Mammalian Cells ◽  
Erk Pathway ◽  
Cavernous Malformations ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Erk Activity ◽  
Hybrid Screening ◽  
Novel Protein

PDCD10 (programmed cell death 10, TFAR15), a novel protein associated with cell apoptosis has been recently implicated in mutations associated with Cerebral Cavernous Malformations (CCM). Yeast two-hybrid screening revealed that PDCD10 interacts with MST4, a member of Ste20-related kinases. This interaction was confirmed by coimmunoprecipitation and colocalization assays in mammalian cells. Furthermore, the co-overexpression of PDCD10 and MST4 promoted cell proliferation and transformation via modulation of the extracellular signal-regulated kinase (ERK) pathway. Potent short interfering RNAs (siRNAs) against PDCD10 (siPDCD10) and MST4 (siMST4) were designed to specifically inhibit the expression of PDCD10 and MST4 mRNA, respectively. The induction of siPDCD10 or siMST4 resulted in decreased expression of endogenous PDCD10 or MST4, which was accompanied by reduced ERK activity and attenuated cell growth and anchorage-independent growth. On the other hand, siMST4 had similar effects in PDCD10-overexpressed cells. And more importantly, we confirmed that either overexpressing or endogenous PDCD10 can increase the MST4 kinase activity in vitro. Our results demonstrated that PDCD10 modulation of ERK signaling was mediated by MST4, and PDCD10 could be a regulatory adaptor necessary for MST4 function, suggesting a link between cerebral cavernous malformation pathogenesis and the ERK-MAPK cascade via PDCD10/MST4.

scholarly journals Differential regulation of the levels of three gap junction mRNAs in Xenopus embryos.

1990 ◽  
Vol 110 (3) ◽  
pp. 597-605 ◽  
Author(s):  
R L Gimlich ◽  
N M Kumar ◽  
N B Gilula
Keyword(s):  
Gap Junction ◽  
Connexin 43 ◽  
Sequence Similarity ◽  
Cdna Libraries ◽  
Amino Acid Sequence Similarity ◽  
Oocyte Growth ◽  
Gap Junction Proteins ◽  
Junction Proteins

Xenopus mRNAs that potentially encode gap junction proteins in the oocyte and early embryo have been identified by low-stringency screening of cDNA libraries with cloned mammalian gap junction cDNAs. The levels of these mRNAs show strikingly different temporal regulation and tissue distribution. Using a nomenclature designed to stress important structural similarities of distinct gap junction gene products, the deduced polypeptides have been designated the Xenopus alpha 1 and alpha 2 gap junction proteins. The alpha 2 gap junction mRNA is a maternal transcript that disappears by the late gastrula stage. It is not detected in any organ of the adult except the ovary, and resides primarily, if not exclusively, in the oocytes and early embryos. The alpha 1 gap junction mRNA appears during organogenesis, and is detected in RNA from a wide variety of organs. It is also found in full-grown oocytes, but is rapidly degraded upon oocyte maturation, both in vivo and in vitro. The alpha 1 and alpha 2 mRNAs encode proteins with different degrees of amino acid sequence similarity to the predominant gap junction subunit of the mammalian heart (connexin 43). Together with our earlier report of a mid-embryonic (beta 1) gap junction mRNA, the results suggest that intercellular communication during oocyte growth and postfertilization development is a complex phenomenon involving the coordinated regulation of several genes.

scholarly journals LncRNA LOC100506178 promotes osteogenic differentiation via regulating miR-214-5p-BMP2 axis in human bone marrow mesenchymal stem cells

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8909
Author(s):  
Lina Li ◽  
Jie Fang ◽  
Yi Liu ◽  
Li Xiao
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Bone Morphogenetic Protein 2 ◽  
Luciferase Reporter ◽  
Alizarin Red ◽  
Dental Implantation

Osteogenic differentiation is an important role in dental implantation. Long no coding RNAs (lncRNAs) are a novel class of noncoding RNAs that have significant effects in a variety of diseases. However, the function and mechanisms of LOC100506178 in osteogenic differentiation and migration of bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation of human bone marrow mesenchymalstem cells (hBMSCs) remain largely unclear. BMP2 was used to induce osteogenic differentiation of hBMSCs. Quantitative real time PCR (qRT-PCR) was used to examine the expression of LOC100506178, miR-214-5p, Runt-related transcription factor 2 (RUNX2), Osterix (Osx), and Alkaline Phosphatase (ALP) in BMP2-induced osteogenic differentiation of hBMSCs. The function of LOC100506178 and miR-214-5p was explored in vitro using Alizarin Red S Staining, ALP activity, as well as in vivo ectopic bone formation. Luciferase reporter assay was performed to assess the association between LOC100506178 and miR-214-5p, as well as miR-214-5p and BMP2. The miR-214-5p sponging potential of LOC100506178 was evaluated by RNA immunoprecipitation. In the present study, the expression of LOC100506178 was found to be increased in BMP2-induced osteogenic differentiation of hBMSCs, accompanied with decreased miR-214-5p expression and increased RUNX2, Osx and ALP expression. LOC100506178 significantly induced, while miR-214-5p suppressed the BMP2-induced osteogenic differentiation of hBMSCs. Mechanistically, LOC100506178 was directly bound to miR-214-5p and miR-214-5p targeted the 3′-untranslated region of BMP2 to negatively regulate its expression. In conclusion, our data indicate a novel molecular pathway LOC100506178/miR-214-5p/BMP2 in relation to hBMSCs differentiation into osteoblasts, which may facilitate bone anabolism.

scholarly journals Signal Pathways Which Promote Invasion and Metastasis: Critical and Distinct Contributions of Extracellular Signal-Regulated Kinase and Ral-Specific Guanine Exchange Factor Pathways

2001 ◽  
Vol 21 (17) ◽  
pp. 5958-5969 ◽  
Author(s):  
Yvona Ward ◽  
Warner Wang ◽  
Elisa Woodhouse ◽  
Ilona Linnoila ◽  
Lance Liotta ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Signal Pathways ◽  
Nucleotide Exchange ◽  
3T3 Cells ◽  
Invasion And Metastasis ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Erk Activity

ABSTRACT Approximately 50% of metastatic tumors contain Ras mutations. Ras proteins can activate at least three downstream signaling cascades mediated by the Raf–MEK–extracellular signal-regulated kinase family, phosphatidylinositol-3 (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (RalGEFs). Here we investigated the contribution of RalGEF and ERK activation to the development of experimental metastasis in vivo and associated invasive properties in vitro. Each pathway contributes distinct properties to the metastatic phenotype. Following lateral tail vein injection, 3T3 cells transformed by constitutively active Raf or MEK produced lung metastasis that displayed circumscribed, noninfiltrating borders. In contrast, 3T3 cells transformed by Ras(12V,37G), a Ras effector mutant that activates RalGEF but not Raf or P13 kinase, formed aggressive, infiltrative metastasis. Dominant negative RalB inhibited Ras(12V,37G)-activated invasion and metastasis, demonstrating the necessity of the RalGEF pathway for a fully transformed phenotype. Moreover, 3T3 cells constitutively expressing a membrane-associated form of RalGEF (RalGDS-CAAX) formed invasive tumors as well, demonstrating that activation of a RalGEF pathway is sufficient to initiate the invasive phenotype. Despite the fact that Ras(12V,37G) expression does not elevate ERK activity, inhibition of this kinase by a conditionally expressed ERK phosphatase demonstrated that ERK activity was necessary for Ras(12V,37G)-transformed cells to express matrix-degrading activity in vitro and tissue invasiveness in vivo. Therefore, these experiments have revealed a hitherto-unknown but essential interaction of the RalGEF and ERK pathways to produce a malignant phenotype. The generality of the role of the RalGEF pathway in metastasis is supported by the finding that Ras(12V,37G) increased the invasiveness of epithelial cells as well as fibroblasts.

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