scholarly journals Modulation of connexin43 alters expression of osteoblastic differentiation markers

2006 ◽  
Vol 290 (4) ◽  
pp. C1248-C1255 ◽  
Author(s):  
Zhongyong Li ◽  
Zhiyi Zhou ◽  
Marnie M. Saunders ◽  
Henry J. Donahue
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Intercellular Communication ◽  
Cell Communication ◽  
Osteoblastic Differentiation ◽  
Type I ◽  
Mrna Levels ◽  
Gap Junctional Intercellular Communication ◽  
Gap Junctional

Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43−). hFOB/Cx43− cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43− cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43− cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor α1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43−. Osteopontin mRNA levels were increased in hFOB/Cx43− relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43− and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells.

Differentiation of human fetal osteoblastic cells and gap junctional intercellular communication

2000 ◽  
Vol 278 (2) ◽  
pp. C315-C322 ◽  
Author(s):  
Henry J. Donahue ◽  
Zhongyong Li ◽  
Zhiyi Zhou ◽  
Clare E. Yellowley
Keyword(s):  
Alkaline Phosphatase ◽  
Gap Junction ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Intercellular Communication ◽  
Connexin 43 ◽  
Osteoblastic Cells ◽  
Gap Junctional Intercellular Communication ◽  
Osteoblastic Cell Line ◽  
Gap Junctional

Gap junctional channels facilitate intercellular communication and in doing so may contribute to cellular differentiation. To test this hypothesis, we examined gap junction expression and function in a temperature-sensitive human fetal osteoblastic cell line (hFOB 1.19) that when cultured at 37°C proliferates rapidly but when cultured at 39.5°C proliferates slowly and displays increased alkaline phosphatase activity and osteocalcin synthesis. We found that hFOB 1.19 cells express abundant connexin 43 (Cx43) protein and mRNA. In contrast, Cx45 mRNA was expressed to a lesser degree, and Cx26 and Cx32 mRNA were not detected. Culturing hFOB 1.19 cells at 39.5°C, relative to 37°C, inhibited proliferation, increased Cx43 mRNA and protein expression, and increased gap junctional intercellular communication (GJIC). Blocking GJIC with 18α-glycyrrhetinic acid prevented the increase in alkaline phosphatase activity resulting from culture at 39.5°C but did not affect osteocalcin levels. These results suggest that gap junction function and expression parallel osteoblastic differentiation and contribute to the expression of alkaline phosphatase activity, a marker for fully differentiated osteoblastic cells.

scholarly journals Cytocompatibility evaluation of hydroxyapatite/collagen composites doped with Zn+2

2007 ◽  
Vol 12 (2) ◽  
pp. 307-312 ◽  
Author(s):  
Maria Helena Santos ◽  
Ana Paula M. Shaimberg ◽  
Patricia Valerio ◽  
Alfredo M. Goes ◽  
Maria de Fátima Leite ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Orthophosphoric Acid ◽  
Type I Collagen ◽  
Enzymatic Digestion ◽  
Osteoblastic Cells ◽  
Type I ◽  
Post Test ◽  
Synthetic Hydroxyapatite

The cytocompatibility of synthetic hydroxyapatite/collagen composites alone or doped with Zn+2 was tested by using primary culture of osteoblasts. The hydroxyapatite (HAP) was synthesized having calcium hydroxide and orthophosphoric acid as precursors. A new HAP composite was developed adding 1.05 w% of Zn(NO3)2.6H2O forming HAPZn. The pure type I collagen (COL) was obtained from bovine pericardium by enzymatic digestion method. The HAP/COL and HAPZn/COL composites were developed and characterized by SEM/EDS. The cell viability and alkaline phosphatase activity in the presence of composites were evaluated by MTT assay and NBT-BCIP assay, respectively, and compared to osteoblastic cells of the control. Three individual experiments were accomplished in triplicates and submitted to the variance analysis and Bonferroni’s post-test with statistically significant at p<0.05. The HAPZn/COL composite did not stimulate the proliferation and increasing of alkaline phosphatase activity of the osteoblastic cells. The tested composites did not alter the cellular viability neither caused alterations in the cellular morphology in 72 h showing adequate properties for biological applications.

scholarly journals Critical Regulation of Bone Morphogenetic Protein-induced Osteoblastic Differentiation by Delta1/Jagged1-activated Notch1 Signaling

2005 ◽  
Vol 280 (16) ◽  
pp. 15842-15848 ◽  
Author(s):  
Masuhiro Nobta ◽  
Tomoo Tsukazaki ◽  
Yasuaki Shibata ◽  
Chang Xin ◽  
Takeshi Moriishi ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Notch Signaling ◽  
Bone Morphogenetic Protein ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Notch Pathway ◽  
Osteoblastic Differentiation ◽  
Differentiation Markers ◽  
Induced Differentiation ◽  
Morphogenetic Protein

Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNA-binding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule andin vivoectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself.

Effects of transforming growth factor-β on normal clonal bone cell populations

1991 ◽  
Vol 69 (2-3) ◽  
pp. 132-140 ◽  
Author(s):  
Rebecca Ber ◽  
Takao Kubota ◽  
Jaro Sodek ◽  
Jane E. Aubin
Keyword(s):  
Alkaline Phosphatase ◽  
Growth Factor ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Transforming Growth Factor ◽  
Bone Cells ◽  
Transforming Growth Factor Β ◽  
Prostaglandin E ◽  
Intracellular Camp ◽  
Type I

Although transforming growth factor-β (TGF-β) has been implicated in the local regulation of bone growth and remodelling, its specific effects on different subpopulations of bone cells have not been elucidated. Cells derived from bone are known to be heterogeneous and include both cells of different lineages and osteoblastic populations with different levels of expression of osteoblast-associated properties. Consequently, we have isolated clonal populations of bone cells to examine more precisely the effects of TGF-β on individual subpopulations. Several clonal populations were isolated by limiting dilution from cells derived from 21-day-old fetal rat calvaria. Two of these clones, RCA 11 and RCB 2, were used here. While the two clones responded similarly to parathyroid hormone (PTH) and isoproterenol (ISP) with increases in intracellular cAMP, prostaglandin E2 (PGE2) elicited a 10-fold higher response in RCB 2 cells compared with RCA 11. RCB 2 cells expressed a 10-fold higher alkaline phosphatase activity compared with RCA 11. Both clones synthesized a variety of bone matrix associated proteins, but only RCA 11 synthesized SPP-1 (osteopontin) constitutively. TGF-β stimulated growth of RCB 2 cells after 24 and 48 h of treatment, but had no effect on growth of RCA 11. TGF-β supported anchorage-independent growth of RCB 2 cells, but not that of RCA 11. A 24-h exposure to TGF-β decreased cAMP responsiveness to PTH and ISP slightly in both clones, but had no effect on PGE2 responses. Significant reductions in alkaline phosphatase activity were seen in both clones after 24- and 48-h treatments with TGF-β. Total protein synthesis as measured by [35S]methionine incorporation was stimulated significantly in both clones, but TGF-β selectively stimulated type I collagen compared with type III collagen. SPARC (osteonectin) and secreted phosphoprotein 1 (SPP-1; osteopontin) were stimulated by TGF-β in both RCA 11 and RCB 2 cells. These results indicate that individual clonal populations of cells within bone may be modulated differentially by TGF-β.Key words: transforming growth factor-β, osteoblasts, clonal cell lines, matrix synthesis.

scholarly journals PDGF-AA promotes cell-to-cell communication in osteocytes through PI3K/Akt signaling pathway

2021 ◽  
Author(s):  
Yang Liu ◽  
Mengmeng Duan ◽  
Daimo Guo ◽  
Shiyi Kan ◽  
L i Zhang ◽  
...  
Keyword(s):  
Intercellular Communication ◽  
Bone Cells ◽  
Bone Matrix ◽  
Cell Communication ◽  
Akt Signaling ◽  
Gap Junctional Intercellular Communication ◽  
Cx43 Expression ◽  
Mineralized Bone Matrix ◽  
Cell Processes ◽  
Gap Junctional

Abstract Osteocytes are the main sensitive cells in bone remodeling due to their potent functional cell processes from the mineralized bone matrix to the bone surface and the bone marrow. Neighboring osteocytes communicate with each other by these cell processes to achieve molecular exchange through gap junction channels. Platelet-derived growth factor-AA (PDGF-AA) has been reported to enhance bone tissue remodeling by promoting cell proliferation, migration, and autocrine secretion in osteoid cell linage. However, the effect of PDGF-AA on intercellular communication between osteocytes is still unclear. In the present study, we elucidated that PDGF-AA could enhance the formation of dendritic processes of osteocytes and the gap junctional intercellular communication by promoting the expression of connexin43 (Cx43). This modulation process was mainly dependent on the activation of phosphorylation of Akt protein by phosphatidylinositol 3-kinase (PI3K)/Akt (also known as protein kinase B, PKB) signaling. Inhibition of PI3K/Akt signaling decreased the Cx43 expression induced by PDGF-AA. These results establish a bridge between PDGF-AA and cell–cell communication in osteocytes, which could help us understand the molecular exchange between bone cells and fracture healing.

IL-6 Induces Osteoblastic Differentiation of Periodontal Ligament Cells

2008 ◽  
Vol 87 (10) ◽  
pp. 937-942 ◽  
Author(s):  
K. Iwasaki ◽  
M. Komaki ◽  
K. Mimori ◽  
E. Leon ◽  
Y. Izumi ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Ascorbic Acid ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Periodontal Ligament ◽  
Kinase Inhibitors ◽  
Osteoblastic Differentiation ◽  
Mitogen Activated Protein Kinase ◽  
Periodontal Ligament Cells ◽  
Igf I

Interleukin (IL)-6 has been considered as an osteolytic factor involved in periodontal disease. However, the function of IL-6 in osteoblastic differentiation of periodontal ligament cells is not clear. We examined the effects of IL-6 and its soluble receptor (sIL-6R) on osteoblastic differentiation of periodontal ligament cells. Osteoblastic differentiation was induced by ascorbic acid. Osteoblast markers, including alkaline phosphatase activity and Runx2 gene expression, were examined. The mechanism of action of IL-6 on osteoblastic differentiation was evaluated by insulin-like growth factor (IGF)-I production and specific inhibitors for the IL-6-signaling molecule. IL-6/sIL-6R enhanced alkaline phosphatase activity and Runx2. Alkaline phosphatase activity was reduced by anti-IGF-I antibody. Mitogen-activated protein kinase and Janus protein tyrosine kinase inhibitors diminished alkaline phosphatase induced by IL-6/sIL-6R. We conclude that IL-6/sIL-6R increases ascorbic-acid-induced alkaline phosphatase activity through IGF-I production, implying that IL-6 acts not only as an osteolytic factor, but also as a mediator of osteoblastic differentiation in periodontal ligament cells.

Inorganic Polyphosphate: a Possible Stimulant of Bone Formation

2007 ◽  
Vol 86 (9) ◽  
pp. 893-897 ◽  
Author(s):  
Y. Hacchou ◽  
T. Uematsu ◽  
O. Ueda ◽  
Y. Usui ◽  
S. Uematsu ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Bone Formation ◽  
Bone Regeneration ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Alveolar Bone ◽  
Bone Mineralization ◽  
Osteoblastic Differentiation ◽  
Inorganic Polyphosphate ◽  
Pit Formation

Inorganic polyphosphates [Poly(P)] are often distributed in osteoblasts. We undertook the present study to verify the hypothesis that Poly(P) stimulates osteoblasts and facilitates bone formation. The osteoblast-like cell line MC 3T3-E1 was cultured with Poly(P), and gene expression and potential mineralization were evaluated by reverse-transcription polymerase chain-reaction. Alkaline phosphatase activity, von Kossa staining, and resorption pit formation analyses were also determined. The potential role of Poly(P) in bone formation was assessed in a rat alveolar bone regeneration model. Poly(P) induced osteopontin, osteocalcin, collagen 1α, and osteoprotegerin expression and increased alkaline phosphatase activity in MC 3T3-E1 cells. Dentin slice pit formation decreased with mouse osteoblast and bone marrow macrophage co-cultivation in the presence of Poly(P). Promotion of alveolar bone regeneration was observed locally in Poly(P)-treated rats. These findings suggest that Poly(P) plays a role in osteoblastic differentiation, activation, and bone mineralization. Thus, local poly(P) delivery may have a therapeutic benefit in periodontal disease.

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