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 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.

Doxorubicin induces EGF receptor-dependent downregulation of gap junctional intercellular communication in rat liver epithelial cells

2005 ◽  
Vol 386 (3) ◽  
pp. 217-223 ◽  
Author(s):  
Kotb Abdelmohsen ◽  
Claudia von Montfort ◽  
Dominik Stuhlmann ◽  
P. Arne Gerber ◽  
Ulrich K.M. Decking ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Rat Liver ◽  
Intercellular Communication ◽  
Connexin 43 ◽  
Egf Receptor ◽  
Gap Junctional Intercellular Communication ◽  
Erk Activation ◽  
Liver Epithelial Cells ◽  
Rat Liver Epithelial Cells ◽  
Gap Junctional

Abstract Exposure of rat liver epithelial cells to doxorubicin, an anthraquinone derivative widely employed in cancer chemotherapy, led to a dose-dependent decrease in gap junctional intercellular communication (GJC). Gap junctions are clusters of inter-cellular channels consisting of connexins, the major connexin in the cells used being connexin-43 (Cx43). Doxorubicin-induced loss of GJC was mediated by activation of extracellular signal-regulated kinase (ERK)-1 and ERK-2, as demonstrated using inhibitors of ERK activation. Furthermore, activation of the epidermal growth factor (EGF) receptor by doxorubicin was responsible for ERK activation and the subsequent attenuation of GJC. Inhibition of GJC, however, was not by direct phosphorylation of Cx43 by ERK-1/2, whereas menadione, a 1,4-naphthoquinone derivative that was previously demonstrated to activate the same EGF receptor-dependent pathway as doxorubicin, resulting in downregulation of GJC, caused strong phos-phorylation of Cx43 at serines 279 and 282. Thus, ERK-dependent downregulation of GJC upon exposure to quinones may occur both by direct phosphorylation of Cx43 and in a phosphorylation-independent manner.

scholarly journals Cancer-Associated Fibroblasts Accelerate Malignant Progression of Non-Small Cell Lung Cancer via Connexin 43-Formed Unidirectional Gap Junctional Intercellular Communication

2018 ◽  
Vol 51 (1) ◽  
pp. 315-336 ◽  
Author(s):  
Min Luo ◽  
Yanmei Luo ◽  
Naiquan Mao ◽  
Guolin Huang ◽  
Cuifang Teng ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Intercellular Communication ◽  
Connexin 43 ◽  
Small Cell ◽  
Migration And Invasion ◽  
Cancer Associated Fibroblasts ◽  
Gap Junctional Intercellular Communication ◽  
Small Cell Lung ◽  
Metabolic Coupling ◽  
Gap Junctional

Background/Aims: Gap junctions, which are assembled by connexins, can directly connect the cytoplasm of adjacent cells and enable gap junctional intercellular communication (GJIC) as well as metabolic coupling between neighboring cells. Here, we investigated the role of connexin 43 (Cx43) and its derived GJIC in the interplay between non-small cell lung cancer (NSCLC) cells and cancer-associated fibroblasts (CAFs). Methods: CAFs and NSCLC cells were co-cultured with direct contact and separated using flow cytometry. Glucose uptake, lactate production, and the expression and activity of PKM-2 and LDH-A in sorted CAFs were measured by a colorimetric assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Meanwhile, E-cadherin and N-cadherin expression and the migration and invasion of sorted NSCLC cells were detected by western blotting, wound width, and Transwell assays. Pyruvate, acetyl-CoA, and citric acid levels, ATP levels, and LDH-B and α-KG activity in sorted NSCLC cells were determined by a colorimetric or fluorometric assay and ELISA, respectively. Functional GJIC between cells and the subcellular location of connexins were detected by a “Parachute” assay and immunofluorescence. Levels of α-SMA, Cx43, and LDH-B in tissue from patients with NSCLC were determined by immunohistochemistry. Results: Cx43 accumulated in the plasma membrane, which favored the assembly of asymmetric unidirectional GJIC from CAFs to NSCLC cells. CAFs underwent increased aerobic glycolysis and promoted the epithelial-mesenchymal transition, migration, and invasion of NSCLC cells. In contrast, NSCLC cells experienced enhanced oxidative phosphorylation upon CAF stimulation, with an increase in ATP generation and thereby activation of the PI3K/Akt and MAPK/ERK pathways. Metabolic coupling between CAFs and NSCLC cells was under the strict control of Cx43-formed unidirectional GJIC. Patients with high tri-expression of α-SMA, Cx43, and LDH-B had the shortest overall survival and relapse-free survival compared with those with individual overexpression or high bi-expression. Conclusion: Cx43-formed unidirectional GJIC plays a critical role in mediating close metabolic cooperation between CAFs and NSCLC cells to support the malignant progression of NSCLC.

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 Effect of glucose concentration in media on alkaline phosphatase activity and mineralization in osteoblastic cells.

1996 ◽  
Vol 71 ◽  
pp. 121
Author(s):  
Yoshitaka Yoshimura ◽  
Yoshiaki Deyama ◽  
Shin-ichi Tanaka ◽  
Kuniaki Suzuki ◽  
Akira Matsumoto
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Glucose Concentration ◽  
Osteoblastic Cells ◽  
Effect Of Glucose
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