scholarly journals Specific functional pathologies of Cx43 mutations associated with oculodentodigital dysplasia

2016 ◽  
Vol 27 (14) ◽  
pp. 2172-2185 ◽  
Author(s):  
John J. Kelly ◽  
Jessica L. Esseltine ◽  
Qing Shao ◽  
Ethylin Wang Jabs ◽  
Jacinda Sampson ◽  
...  
Keyword(s):  
Gap Junction ◽  
Dermal Fibroblast ◽  
Phenotypic Variability ◽  
Human Dermal Fibroblast ◽  
Cell Polarization ◽  
Gap Junctional Intercellular Communication ◽  
Multiple Organs ◽  
Clinical Presentations ◽  
Gap Junctional ◽  
Oculodentodigital Dysplasia

Oculodentodigital dysplasia (ODDD) is a rare genetic disease that affects the development of multiple organs in the human body. More than 70 mutations in the gap junction connexin43 (Cx43) gene, GJA1, are associated with ODDD, most of which are inherited in an autosomal dominant manner. Many patients exhibit similar clinical presentations. However, there is high intrafamilial and interfamilial phenotypic variability. To better understand this variability, we established primary human dermal fibroblast cultures from several ODDD patients and unaffected controls. In the present study, we characterized three fibroblast lines expressing heterozygous p.L7V, p.G138R, and p.G143S Cx43 variants. All ODDD fibroblasts exhibited slower growth, reduced migration, and defective cell polarization, traits common to all ODDD fibroblasts studied so far. However, we found striking differences in overall expression levels, with p.L7V down-regulated at the mRNA and protein level. Although all of the Cx43 variants could traffic to the cell surface, there were stark differences in gap junction plaque formation, gap junctional intercellular communication, Cx43 phosphorylation, and hemichannel activity among Cx43 variants, as well as subtle differences in myofibroblast differentiation. Together these findings enabled us to discover mutation-specific pathologies that may help to predict future clinical outcomes.

scholarly journals High-throughput measurement of gap junctional intercellular communication

2014 ◽  
Vol 306 (12) ◽  
pp. H1708-H1713 ◽  
Author(s):  
Jun Liu ◽  
Vinayakumar Siragam ◽  
Jun Chen ◽  
Michael D. Fridman ◽  
Robert M. Hamilton ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Cell Lines ◽  
High Throughput ◽  
Intercellular Communication ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Cell Injection ◽  
Operation Speed ◽  
Gap Junctional

Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.

Gap junctions and tumour progression

2002 ◽  
Vol 80 (2) ◽  
pp. 136-141 ◽  
Author(s):  
Christian CG Naus
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Tumour Progression ◽  
Second Messengers ◽  
Regulation Of Gene Expression ◽  
Growth Control ◽  
Gap Junctional Intercellular Communication ◽  
Gap Junction Channels ◽  
Junctional Coupling ◽  
Gap Junctional

Gap junctional intercellular communication has been implicated in growth control and differentiation. The mechanisms by which connexins, the gap junction proteins, act as tumor suppressors are unclear. In this review, several different mechanisms are considered. Since transformation results in a loss of the differentiated state, one mechanism by which gap junctions may control tumour progression is to promote or enhance differentiation. Processes of differentiation and growth control are mediated at the genetic level. Thus, an alternative or complimentary mechanism of tumour suppression could involve the regulation of gene expression by connexins and gap junctional coupling. Finally, gap junction channels form a conduit between cells for the exchange of ions, second messengers, and small metabolites. It is clear that the sharing of these molecules can be rather selective and may be involved in growth control processes. In this review, examples will be discussed that provide evidence for each of these mechanisms. Taken together, these findings point to a variety of mechanims by which connexins and the gap junction channels that they form may control tumour progression.Key words: gap junctions, connexin, cancer.

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 The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins.

1991 ◽  
Vol 11 (10) ◽  
pp. 5364-5371 ◽  
Author(s):  
J L Brissette ◽  
N M Kumar ◽  
N B Gilula ◽  
G P Dotto
Keyword(s):  
Gap Junction ◽  
Intercellular Communication ◽  
Molecular Mechanisms ◽  
Serine Phosphorylation ◽  
Tumor Promoter ◽  
Gap Junctional Intercellular Communication ◽  
Beta 2 ◽  
Gap Junction Proteins ◽  
Gap Junctional ◽  
Junction Proteins

Gap junctional intercellular communication is inhibited in response to tumor promoters and oncogene transformation, suggesting that loss of this function is an important step in tumor formation. To elucidate the molecular mechanisms responsible for this inhibition, we examined the expression of gap junction proteins and mRNA in mouse primary keratinocytes after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or ras transformation. During normal cell growth, keratinocytes expression the alpha 1 (connexin 43) and beta 2 (connexin 26) proteins. Within 5 min of TPA treatment, the alpha 1 protein became rapidly phosphorylated on serine residues and its expression was dramatically reduced by 24 h. The beta 2 protein, after an initial increase in expression, was also significantly reduced 24 h after treatment with TPA. ras transformation caused changes similar to those induced by TPA. The alpha 1 protein underwent an increase in serine phosphorylation, although its expression declined only slightly, while beta 2 expression was greatly reduced. The effects of TPA and ras on alpha 1 expression were additive; treatment of ras-transformed cells with TPA resulted in increased alpha 1 phosphorylation, with greatly decreased protein levels, much lower than those generated by either agent alone. These data provide a likely explanation for the similar and synergistic inhibition of gap junctional intercellular communication by phorbol esters and ras.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins

1991 ◽  
Vol 11 (10) ◽  
pp. 5364-5371
Author(s):  
J L Brissette ◽  
N M Kumar ◽  
N B Gilula ◽  
G P Dotto
Keyword(s):  
Gap Junction ◽  
Intercellular Communication ◽  
Molecular Mechanisms ◽  
Serine Phosphorylation ◽  
Tumor Promoter ◽  
Gap Junctional Intercellular Communication ◽  
Beta 2 ◽  
Gap Junction Proteins ◽  
Gap Junctional ◽  
Junction Proteins

Gap junctional intercellular communication is inhibited in response to tumor promoters and oncogene transformation, suggesting that loss of this function is an important step in tumor formation. To elucidate the molecular mechanisms responsible for this inhibition, we examined the expression of gap junction proteins and mRNA in mouse primary keratinocytes after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or ras transformation. During normal cell growth, keratinocytes expression the alpha 1 (connexin 43) and beta 2 (connexin 26) proteins. Within 5 min of TPA treatment, the alpha 1 protein became rapidly phosphorylated on serine residues and its expression was dramatically reduced by 24 h. The beta 2 protein, after an initial increase in expression, was also significantly reduced 24 h after treatment with TPA. ras transformation caused changes similar to those induced by TPA. The alpha 1 protein underwent an increase in serine phosphorylation, although its expression declined only slightly, while beta 2 expression was greatly reduced. The effects of TPA and ras on alpha 1 expression were additive; treatment of ras-transformed cells with TPA resulted in increased alpha 1 phosphorylation, with greatly decreased protein levels, much lower than those generated by either agent alone. These data provide a likely explanation for the similar and synergistic inhibition of gap junctional intercellular communication by phorbol esters and ras.

A germline-specific gap junction protein required for survival of differentiating early germ cells

Development ◽  
2002 ◽  
Vol 129 (10) ◽  
pp. 2529-2539 ◽  
Author(s):  
Salli I. Tazuke ◽  
Cordula Schulz ◽  
Lilach Gilboa ◽  
Mignon Fogarty ◽  
Anthony P. Mahowald ◽  
...  
Keyword(s):  
Germ Cell ◽  
Gap Junction ◽  
Germ Cells ◽  
Somatic Cells ◽  
Gap Junctional Intercellular Communication ◽  
Germ Cell Differentiation ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
Zero Population Growth ◽  
Gap Junctional

Germ cells require intimate associations and signals from the surrounding somatic cells throughout gametogenesis. The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Animals with a null mutation in zpg are viable but sterile and have tiny gonads. Adult zpg-null gonads contain small numbers of early germ cells, resembling stem cells or early spermatogonia or oogonia, but lack later stages of germ cell differentiation. In the male, Zpg protein localizes to the surface of spermatogonia, primarily on the sides adjacent to the somatic cyst cells. In the female, Zpg protein localizes to germ cell surfaces, both those adjacent to surrounding somatic cells and those adjacent to other germ cells. We propose that Zpg-containing gap junctional hemichannels in the germ cell plasma membrane may connect with hemichannels made of other innexin isoforms on adjacent somatic cells. Gap junctional intercellular communication via these channels may mediate passage of crucial small molecules or signals between germline and somatic support cells required for survival and differentiation of early germ cells in both sexes.

Effects of cadmium on gap junctional intercellular communication in WB-F344 rat liver epithelial cells

2001 ◽  
Vol 20 (11) ◽  
pp. 577-583 ◽  
Author(s):  
S-H Jeong ◽  
M-H Cho ◽  
J-H Cho
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cells ◽  
Gap Junctions ◽  
Cell Viability ◽  
Gap Junction ◽  
Intercellular Communication ◽  
Tumor Promotion ◽  
Gap Junctional Intercellular Communication ◽  
Rat Liver Epithelial Cells ◽  
Gap Junctional

Cadmium has been associated with a number of tumors but its role in tumor promotion has not been elucidated clearly or the results obtained from various studies have been conflicting. This study was designed to investigate the effects of cadmium on the gap junctional intercellular communication (GJIC), number of gap junctions per cell, and cell proliferation in WB-F344 rat liver epithelial cells from the viewpoint of tumor promotion. GJIC was monitored by counting the cells stained with Lucifer yellow CH dye, using the scrape-loading and dye-transfer method. The numbers of gap junctions per cell were visually quantitated after an indirect immunostaining for gap junction protein using an antibody to connexin 43. Cell proliferation was assayed by direct counting of the living cells using the trypan blue dye exclusion method. In the time course study, cells treated with 200 μM CdCl2 showed rapid and nearly complete inhibition of GJIC (approximately 14% of the control) and a decrease in the number of gap junctions per cell (approximately 21% of the control) at 30 min, and the decrease continued up to 4 h without any changes in the cell viability. Treatment with CdCl2 7.4-200 μM) for 4 h resulted in the decrease of GJIC and gap junction numbers per cell in a dose-response pattern without changes in the cell viability. In the long-term (14 days) exposure studies at doses of 0.01-7.4 μM CdCl2, an increase in cell proliferation was observed at low doses of 0.03-2.5 μM CdCl2, with GJIC also decreasing. These data demonstrate that cadmium inhibits GJIC, reduces the number of gap junctions per cell, and induces cell proliferation while decreasing the function of the gap junction.

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