scholarly journals Effects of Alpha-Connexin Carboxyl-Terminal Peptide (aCT1) and Bowman-Birk Protease Inhibitor (BBI) on Canine Oral Mucosal Melanoma (OMM) Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Ayami Sato ◽  
Ivone Izabel Mackowiak da Fonseca ◽  
Márcia Kazumi Nagamine ◽  
Gabriela Fernandes de Toledo ◽  
Rennan Olio ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Protease Inhibitor ◽  
Gap Junctions ◽  
Cell Viability ◽  
Cancer Cells ◽  
Cell Communication ◽  
Cx43 Expression ◽  
Altered Expression ◽  
Junction Protein ◽  
Carboxyl Terminal

Oral mucosal melanomas (OMM) are aggressive cancers in dogs, and are good models for human OMM. Gap junctions are composed of connexin units, which may have altered expression patterns and/or subcellular localization in cancer cells. Cell-to-cell communication by gap junctions is often impaired in cancer cells, including in melanomas. Meanwhile, the upregulated expression of the gap junction protein connexin 43 (Cx43) inhibits melanoma progression. The α-connexin carboxyl-terminal (aCT1) peptide reportedly maintains Cx43 expression and cell-cell communication in human mammary cells and increases the communication activity through gap junctions in functional assays, therefore causing decreased cell proliferation. The Bowman-Birk protease inhibitor (BBI), a component of soybeans, induces Cx43 expression in several tumor cells as a trypsin–chymotrypsin inhibition function, with antineoplastic effects. This study investigated the effect of aCT1 peptide and BBI treatment, alone or in combination, on TLM1 canine melanoma cell viability. Cell viability after treatment with aCT1, the reverse sequence peptide (R-pep), and/or BBI for 5 days was analyzed by PrestoBlue assay. Immunofluorescence was used to observe Cx43 localization and expression. aCT1 (200 μM) alone did not significantly decrease cell viability in TLM1 cells, whereas BBI (400 μg/ml) alone significantly decreased the TLM1 viability. Combined treatment with both aCT1 (200 μM) and BBI (400 μg/ml) significantly decreased cell viability in TLM1 cells. Cx43 expression, as identified by immunostainings in TLM1 cells, was increased in the cell membrane after the combination treatment with BBI and aCT1. This dual treatment can be combined to achieve the anticancer activity, possibly by increasing Cx 43 expression and affecting Cx43 migration to the cell membrane. In conclusion, a treatment strategy targeting Cx43 with BBI and aCT1 may possibly lead to new effective therapies for canine OMM.

Analysis of distribution patterns of gap junctions during development of embryonic chick facial primordia and brain

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 509-522
Author(s):  
R. Minkoff ◽  
S.B. Parker ◽  
E.L. Hertzberg
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Uniform Distribution ◽  
Rat Liver ◽  
Connexin 43 ◽  
Cell Communication ◽  
Exterior Surface ◽  
Junction Protein ◽  
Primary Palate ◽  
Gap Junction Protein

Gap junction distribution in the facial primordia of chick embryos at the time of primary palate formation was studied employing indirect immunofluorescence localization with antibodies to gap junction proteins initially identified in rat liver (27 × 10(3) Mr, connexin 32) and heart (43 × 10(3) Mr, connexin 43). Immunolocalization with antibodies to the rat liver gap junction protein (27 × 10(3) Mr) demonstrated a ubiquitous and uniform distribution in all regions of the epithelium and mesenchyme except the nasal placode. In the placodal epithelium, a unique non-random distribution was found characterized by two zones: a very heavy concentration of signal in the superficial layer of cells adjacent to the exterior surface and a region devoid of detectable signal in the interior cell layer adjacent to the mesenchyme. This pattern was seen during all stages of placode invagination that were examined. The separation of gap junctions in distinct cell layers was unique to the nasal placode, and was not found in any other region of the developing primary palate. One other tissue was found that exhibited this pattern-the developing neural epithelium of the brain and retina. These observations suggest the presence of region-specific signaling mechanisms and, possibly, an impedance of cell communication among subpopulations of cells in these structures at critical stages of development. Immunolocalization with antibodies to the ‘heart’ 43 × 10(3) Mr gap junction protein also revealed the presence of gap junction protein in facial primordia and neural epithelium. A non-uniform distribution of immunoreactivity was also observed for connexin 43.

scholarly journals Cx43 and Associated Cell Signaling Pathways Regulate Tunneling Nanotubes in Breast Cancer Cells

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2798
Author(s):  
Alexander Tishchenko ◽  
Daniel D. Azorín ◽  
Laia Vidal-Brime ◽  
María José Muñoz ◽  
Pol Jiménez Arenas ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gap Junctions ◽  
Small Molecules ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Connexin 43 ◽  
Breast Cancer Cell Lines ◽  
Tunneling Nanotubes ◽  
Cx43 Expression ◽  
Cancer Signaling

Connexin 43 (Cx43) forms gap junctions that mediate the direct intercellular diffusion of ions and small molecules between adjacent cells. Cx43 displays both pro- and anti-tumorigenic properties, but the mechanisms underlying these characteristics are not fully understood. Tunneling nanotubes (TNTs) are long and thin membrane projections that connect cells, facilitating the exchange of not only small molecules, but also larger proteins, organelles, bacteria, and viruses. Typically, TNTs exhibit increased formation under conditions of cellular stress and are more prominent in cancer cells, where they are generally thought to be pro-metastatic and to provide growth and survival advantages. Cx43 has been described in TNTs, where it is thought to regulate small molecule diffusion through gap junctions. Here, we developed a high-fidelity CRISPR/Cas9 system to knockout (KO) Cx43. We found that the loss of Cx43 expression was associated with significantly reduced TNT length and number in breast cancer cell lines. Notably, secreted factors present in conditioned medium stimulated TNTs more potently when derived from Cx43-expressing cells than from KO cells. Moreover, TNT formation was significantly induced by the inhibition of several key cancer signaling pathways that both regulate Cx43 and are regulated by Cx43, including RhoA kinase (ROCK), protein kinase A (PKA), focal adhesion kinase (FAK), and p38. Intriguingly, the drug-induced stimulation of TNTs was more potent in Cx43 KO cells than in wild-type (WT) cells. In conclusion, this work describes a novel non-canonical role for Cx43 in regulating TNTs, identifies key cancer signaling pathways that regulate TNTs in this setting, and provides mechanistic insight into a pro-tumorigenic role of Cx43 in cancer.

scholarly journals Antitumor Effect of IL-2 and TRAIL Proteins Expressed by Recombinant Salmonella in Murine Bladder Cancer Cells

2021 ◽  
Vol 55 (4) ◽  
pp. 460-476
Keyword(s):  
Oxidative Stress ◽  
Bladder Cancer ◽  
Cell Membrane ◽  
Cell Viability ◽  
Cancer Cells ◽  
Cell Morphology ◽  
Antitumor Effect ◽  
Interleukin 2 ◽  
Continuous Increase ◽  
Bladder Cancer Cells

Background/Aims: Cancer is the second most deadly disease in the world. The bladder cancer is one of the most aggressive types and shows a continuous increase in the number of cases. The use of bacteria as live vectors to deliver molecules directly to the tumor is a promising tool and has been used as an adjuvant treatment against several types of cancer. The aim of this study was to investigate the antitumor effect of Interleukin 2 (IL-2), TNF-related apoptosis-inducing ligand (TRAIL) and protein MIX against murine bladder cancer cells, lineage MB49. Methods: The attenuated Salmonella strain SL3261 was transformed by inserting the IL-2 and TRAIL genes. The effects of proteins on cell viability (MTT method), cell morphology (optical microscopy), cell recovery (clonogenic assay), cell membrane (lactate dehydrogenase release - LDH), on oxidative stress pathway (levels of nitric oxide, NO) and apoptosis (flow cytometry and high resolution epifluorescence images) were evaluated at intervals of 24 and 48 hours of action. Results: The results showed that there was a decrease in cell viability via damage to the cell membrane, alteration of cell morphology, non-recovery of cells, increase in the production of NO and incubate for of cells in the state of apoptosis in the two periods analyzed. Conclusion: The data presented suggest that IL-2, TRAIL and their MIX proteins in MB49 cells have cytotoxic potential and that this is associated with oxidative stress and apoptosis pathways. These results may contribute to the development of new therapeutic strategies for bladder cancer.

scholarly journals Amyloid-β regulates gap junction protein connexin 43 trafficking in cultured primary astrocytes

2020 ◽  
Vol 295 (44) ◽  
pp. 15097-15111
Author(s):  
Mahua Maulik ◽  
Lakshmy Vasan ◽  
Abhishek Bose ◽  
Saikat Dutta Chowdhury ◽  
Neelanjana Sengupta ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
Cx43 Expression ◽  
Chemical Chaperone ◽  
Junction Protein ◽  
Gap Junction Coupling ◽  
Gap Junction Protein ◽  
Junction Coupling ◽  
And Function

Altered expression and function of astroglial gap junction protein connexin 43 (Cx43) has increasingly been associated to neurotoxicity in Alzheimer disease (AD). Although earlier studies have examined the effect of increased β-amyloid (Aβ) on Cx43 expression and function leading to neuronal damage, underlying mechanisms by which Aβ modulates Cx43 in astrocytes remain elusive. Here, using mouse primary astrocyte cultures, we have examined the cellular processes by which Aβ can alter Cx43 gap junctions. We show that Aβ25-35 impairs functional gap junction coupling yet increases hemichannel activity. Interestingly, Aβ25-35 increased the intracellular pool of Cx43 with a parallel decrease in gap junction assembly at the surface. Intracellular Cx43 was found to be partly retained in the endoplasmic reticulum-associated cell compartments. However, forward trafficking of the newly synthesized Cx43 that already reached the Golgi was not affected in Aβ25-35-exposed astrocytes. Supporting this, treatment with 4-phenylbutyrate, a well-known chemical chaperone that improves trafficking of several transmembrane proteins, restored Aβ-induced impaired gap junction coupling between astrocytes. We further show that interruption of Cx43 endocytosis in Aβ25-35-exposed astrocytes resulted in their retention at the cell surface in the form of functional gap junctions indicating that Aβ25-35 causes rapid internalization of Cx43 gap junctions. Additionally, in silico molecular docking suggests that Aβ can bind favorably to Cx43. Our study thus provides novel insights into the cellular mechanisms by which Aβ modulates Cx43 function in astrocytes, the basic understanding of which is vital for the development of alternative therapeutic strategy targeting connexin channels in AD.

scholarly journals Cx43 and Associated Cell Signaling Pathways Regulate Tunneling Nanotubes in Breast Cancer Cells

2020 ◽  
Author(s):  
Alexander Tishchenko ◽  
Daniel Domínguez Azorín ◽  
Laia Vidal-Brime ◽  
María José Muñoz ◽  
Pol Jiménez Arenas ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gap Junctions ◽  
Small Molecules ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Connexin 43 ◽  
Breast Cancer Cell Lines ◽  
Tunneling Nanotubes ◽  
Cx43 Expression ◽  
Cancer Signaling

Connexin 43 (Cx43) forms gap junctions that mediate the direct intercellular diffusion of ions and small molecules between adjacent cells. Cx43 displays both pro- and anti-tumorigenic properties, but the mechanisms underlying these characteristics are not fully understood. Tunneling nanotubes (TNTs) are long and thin membrane projections that connect cells, facilitating the exchange of not only small molecules, but also larger proteins, organelles, bacteria, and viruses. Typically, TNTs exhibit increased formation under conditions of cellular stress and are more prominent in cancer cells, where they are generally thought to be pro-metastatic and to provide growth and survival advantages. Cx43 has been described in TNTs, where it is thought to regulate small molecule diffusion through gap junctions. Here, we developed a high-fidelity CRISPR/Cas9 system to knockout (KO) Cx43. We found that loss of Cx43 expression was associated with significantly reduced TNT length and number in breast cancer cell lines. Notably, secreted factors present in conditioned medium stimulated TNTs more potently when derived from Cx43-expressing cells than from KO cells. Moreover, TNT formation was significantly induced by inhibition of several key cancer signaling pathways that both regulate Cx43 and are regulated by Cx43, including RhoA kinase (ROCK), protein kinase A (PKA), focal adhesion kinase (FAK), and p38. Intriguingly, drug-induced stimulation of TNTs was more potent in Cx43 KO cells than in wild-type cells. In conclusion, this work describes a novel non-canonical role for Cx43 in regulating TNTs, identifies key cancer signaling pathways that regulate TNTs in this setting, and provides mechanistic insight into a pro-tumorigenic role of Cx43 in cancer.

scholarly journals Glucose-evoked alterations in connexin43-mediated cell-to-cell communication in human collecting duct: a possible role in diabetic nephropathy

2006 ◽  
Vol 291 (5) ◽  
pp. F1045-F1051 ◽  
Author(s):  
Claire E. Hills ◽  
Rosemary Bland ◽  
Dianne C. Wheelans ◽  
Jeanette Bennett ◽  
Pierre M. Ronco ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Gap Junctions ◽  
High Glucose ◽  
Transient Receptor Potential ◽  
Lucifer Yellow ◽  
Transmission Rate ◽  
Collecting Duct ◽  
Cell Communication ◽  
Sodium Absorption ◽  
Cx43 Expression

Aberrant sodium absorption has been linked to the development of hypertension in both renal disease and diabetes. Efficient absorption depends on coordination of cellular activity across the entire epithelium via cell-to-cell coupling. In the current study we have utilized a model human collecting duct cell line (HCD) to assess the role of connexin43 (Cx43)-mediated gap junctions in the transfer of intracellular Ca2+ transients within coupled cell clusters. HCD cells express Cx43 mRNA and protein, as well as that for the mechanosensitive transient receptor potential receptor (TRPV4). Mechanical stimulation of individual cells within a cluster evoked a transient rise in cytosolic Ca2+ concentration ([Ca2+]i) that propagated between cells via a heptanol-sensitive mechanism. The rise in [Ca2+]i was dependent on both store release and Ca2+-influx pathways. Lucifer yellow dye transfer and Cx43 knockdown experiments confirmed direct cell-to-cell communication. Application of the Ca2+ ionophore ionomycin, or an increase in glucose (5 to 25 mM), produced a time-dependent (48 h) increase in Cx43 protein expression. The transmission rate of touch-evoked Ca2+ transients between coupled cells was accelerated after exposure to high glucose, providing a functional correlate to increased Cx43 expression. These data suggest a pivotal role for Cx43-mediated gap junctions in the synchronization of activity between HCD cells in response to stimuli that mimic osmotic and physical changes. Cx43 expression and cell-to-cell communication increased in response to high glucose and may protect the collecting duct from renal damage associated with more established diabetic nephropathy.

scholarly journals Changes in Gap Junction Distribution and Connexin Expression Pattern During Human Fetal Skin Development

2002 ◽  
Vol 50 (11) ◽  
pp. 1493-1500 ◽  
Author(s):  
Ken Arita ◽  
Masashi Akiyama ◽  
Yukiko Tsuji ◽  
James R. McMillan ◽  
Robin A.J. Eady ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Basal Layer ◽  
Cell Communication ◽  
Fetal Skin ◽  
Typical Structure ◽  
Cx43 Expression ◽  
Skin Development ◽  
Connexin Expression ◽  
Epidermal Development

Gap junctions are intercellular channels composed of connexin subunits that mediate cell-cell communication. The functions of gap junctions are believed to be associated with cell proliferation and differentiation and to be important in maintaining tissue homeostasis. We therefore investigated the expression of connexins (Cx)26 and 43, the two major connexins in human epidermis, and examined the formation of gap junctions during human fetal epidermal development. By immunofluorescence, Cx26 expression was observed between 49 and 96 days’ estimated gestational age (EGA) but was not present from 108 days’ EGA onwards. Conversely, Cx43 expression was observed from 88 days’ EGA onwards. Using electron microscopy, the typical structure of gap junctions was observed from 120 days’ EGA. The number of gap junctions increased over time and they were more common in the upper layers, within the periderm and intermediate keratinocyte layers rather than the basal layer. Immunoelectron microscopy revealed Cx43 labeling on the gap junction structures after 105 days’ EGA. Formation of gap junctions increased as skin developed, suggesting that gap junctions may play an important role in fetal skin development. Furthermore, the changing patterns of connexin expression suggest that Cx26 is important for early fetal epidermal development.

scholarly journals Luteinizing Hormone-Induced Connexin 43 Down-Regulation: Inhibition of Translation

Endocrinology ◽  
2004 ◽  
Vol 145 (4) ◽  
pp. 1617-1624 ◽  
Author(s):  
Yael Kalma ◽  
Irit Granot ◽  
Dalia Galiani ◽  
Amihai Barash ◽  
Nava Dekel
Keyword(s):  
Connexin 43 ◽  
Ovarian Follicle ◽  
Cell Communication ◽  
Signal Transduction Pathway ◽  
Mapk Signaling ◽  
Down Regulation ◽  
Cx43 Expression ◽  
Mapk Cascades ◽  
Junction Protein

Abstract The coordinated function of the different compartments of the follicle, the oocyte and the somatic cumulus/granulosa cells, is enabled by the presence of a network of cell-to-cell communication generated by gap junctions. Connexin 43 (Cx43) is the most abundant gap junction protein expressed by the ovarian follicle. The expression of Cx43 is subjected to the control of gonadotropins as follows: FSH up-regulates, whereas LH down-regulates its levels. The aim of this study was to explore the mechanism by which LH reduces the levels of Cx43 and to identify the signal transduction pathway involved in this process. The effect of LH was studied in vitro using isolated intact ovarian follicles. The possible mediators of LH-induced Cx43 down-regulation were examined by incubating the follicles with LH in the presence or absence of inhibitors of protein kinase A (PKA) and of MAPK signaling pathways. Our experiments revealed a 3-h half-life of Cx43 in both control and LH-treated follicles, suggesting that LH did not affect the rate of Cx43 degradation. We further demonstrated that the level of Cx43 mRNA was not significantly influenced by this gonadotropin. However, upon LH administration, [35S]methionine incorporation into Cx43 protein was remarkably reduced. The LH-induced arrest of Cx43 synthesis was counteracted by inhibitors of both the PKA and the MAPK cascades. We show herein that LH inhibits Cx43 expression by reducing its rate of translation and that this effect is mediated by both PKA and MAPK.

scholarly journals Estradiol Reduces Connexin43 Gap Junctions in the Uterus during Adenomyosis in Cows

2016 ◽  
Vol 19 (3) ◽  
pp. 609-617 ◽  
Author(s):  
A.J. Korzekwa ◽  
M. Łupicka ◽  
B.M. Socha ◽  
A.A. Szczepańska
Keyword(s):  
Protein Expression ◽  
Gap Junctions ◽  
Mrna Expression ◽  
Uterine Tissue ◽  
Cx43 Expression ◽  
Junction Protein ◽  
Uterine Adenomyosis ◽  
Mrna And Protein Expression ◽  
Gap Junction Protein ◽  
Cx43 Mrna

Abstract Adenomyosis is defined as the presence of glandular foci external to the endometrium of the uterus, either in the myometrium or/and perimetrium, depending on the progress of this dysfunction. To date, we showed that steroids secretion and prolactin expression and proliferative processes are disturbed during uterine adenomyosis in cows. During endometriosis in eutopic endometrium in women, gap junctions are down regulated. The transmembrane gap junction protein, connexin (Cx43) is necessary for endometrial morphological, biochemical and angiogenic functions. The aim of this study is recognition of adenomyosis etiology by determination of the role of Cx43 in this process. Immunolocalization and comparison of Cx43 mRNA and protein expression in healthy (N=9) and adenomyotic uterine tissue (N=9), and Cx43 mRNA expression (real time PCR) in uterine stromal – myometrium co-culture under 24-hour stimulation with 17-beta estradiol (10−7M) isolated from healthy (N=5) and adenomyotic (N=5) cows were determined. Cx43 was localized in healthy and adenomyotic uteri. mRNA and protein expression was down-regulated in uterine tissue in adenomyotic compared with healthy cows (p<0.05). Estradiol stimulated Cx43 mRNA expression in myometrial cell culture and co-culture of stromal and myometrial cells in adenomyotic compared with healthy cows (p<0.05). In summary, down-regulation of Cx43 expression in the junction zone might play an important role in pathogenesis of adenomyosis. Estradiol modulates gap junctions during adenomyosis.

scholarly journals The tight junction protein CAR regulates cardiac conduction and cell–cell communication

2008 ◽  
Vol 205 (10) ◽  
pp. 2369-2379 ◽  
Author(s):  
Ulrike Lisewski ◽  
Yu Shi ◽  
Uta Wrackmeyer ◽  
Robert Fischer ◽  
Chen Chen ◽  
...  
Keyword(s):  
Heart Disease ◽  
Tight Junction ◽  
Cardiac Remodeling ◽  
Cell Communication ◽  
Underlying Mechanism ◽  
Cardiac Conduction ◽  
Altered Expression ◽  
Progressive Loss ◽  
Junction Protein ◽  
Adenovirus Receptor

The Coxsackievirus-adenovirus receptor (CAR) is known for its role in virus uptake and as a protein of the tight junction. It is predominantly expressed in the developing brain and heart and reinduced upon cardiac remodeling in heart disease. So far, the physiological functions of CAR in the adult heart are largely unknown. We have generated a heart-specific inducible CAR knockout (KO) and found impaired electrical conduction between atrium and ventricle that increased with progressive loss of CAR. The underlying mechanism relates to the cross talk of tight and gap junctions with altered expression and localization of connexins that affect communication between CAR KO cardiomyocytes. Our results indicate that CAR is not only relevant for virus uptake and cardiac remodeling but also has a previously unknown function in the propagation of excitation from the atrium to the ventricle that could explain the association of arrhythmia and Coxsackievirus infection of the heart.

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