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15 pages, 2779 KiB

Open AccessArticle

Connexin43 Is Required for the Effective Activation of Spleen Cells and Immunoglobulin Production

by Yanru Huang, Zhimin Mao, Xiling Zhang, Xiawen Yang, Norifumi Sawada, Masayuki Takeda and Jian Yao

Int. J. Mol. Sci. 2019, 20(22), 5789; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20225789 - 18 Nov 2019

Cited by 7 | Viewed by 2738

Abstract

Gap junctions (Gjs), formed by specific protein termed connexins (Cxs), regulate many important cellular processes in cellular immunity. However, little is known about their effects on humoral immunity. Here we tested whether and how Gj protein connexin43 (Cx43) affected antibody production in spleen [...] Read more.

Gap junctions (Gjs), formed by specific protein termed connexins (Cxs), regulate many important cellular processes in cellular immunity. However, little is known about their effects on humoral immunity. Here we tested whether and how Gj protein connexin43 (Cx43) affected antibody production in spleen cells. Detection of IgG in mouse tissues and serum revealed that wild-type (Cx43^+/+) mouse had a significantly higher level of IgG than Cx43 heterozygous (Cx43^+/−) mouse. Consistently, spleen cells from Cx43^+/+ mouse produced more IgG under both basal and lipopolysaccharide (LPS)-stimulated conditions. Further analysis showed that LPS induced a more dramatic activation of ERK and cell proliferation in Cx43^+/+ spleen cells, which was associated with a higher pro-oxidative state, as indicated by the increased NADPH oxidase 2 (NOX2), TXNIP, p38 activation and protein carbonylation. In support of a role of the oxidative state in the control of lymphocyte activation, exposure of spleen cells to exogenous superoxide induced Cx43 expression, p38 activation and IgG production. On the contrary, inhibition of NOX attenuated the effects of LPS. Collectively, our study characterized Cx43 as a novel molecule involved in the control of spleen cell activation and IgG production. Targeting Cx43 could be developed to treat certain antibody-related immune diseases. Full article

(This article belongs to the Special Issue Connexin-Based Channels in Inflammatory Processes)

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16 pages, 4280 KiB

Open AccessArticle

Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing

by Francisca Hofmann, Mariela Navarrete, Javiera Álvarez, Israel Guerrero, María Alejandra Gleisner, Andrés Tittarelli and Flavio Salazar-Onfray

Int. J. Mol. Sci. 2019, 20(18), 4509; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20184509 - 12 Sep 2019

Cited by 25 | Viewed by 4973

Abstract

Upon tumor antigen recognition, cytotoxic T lymphocytes (CTLs) and target cells form specialized supramolecular structures, called cytotoxic immunological synapses, which are required for polarized delivery of cytotoxic granules. In previous reports, we described the accumulation of connexin 43 (Cx43)-formed gap junctions (GJs) at [...] Read more.

Upon tumor antigen recognition, cytotoxic T lymphocytes (CTLs) and target cells form specialized supramolecular structures, called cytotoxic immunological synapses, which are required for polarized delivery of cytotoxic granules. In previous reports, we described the accumulation of connexin 43 (Cx43)-formed gap junctions (GJs) at natural killer (NK) cell–tumor cell cytotoxic immunological synapse. In this report, we demonstrate the functional role of Cx43-GJs at the cytotoxic immunological synapse established between CTLs and melanoma cells during cytotoxicity. Using confocal microscopy, we evaluated Cx43 polarization to the contact site between CTLs isolated from pMEL-1 mice and B16F10 melanoma cells. We knocked down Cx43 expression in B16F10 cells and evaluated its role in the formation of functional GJs and the cytotoxic activity of CTLs, by calcein transfer and granzyme B activity assays, respectively. We found that Cx43 localizes at CTL/B16F10 intercellular contact sites via an antigen-dependent process. We also found that pMEL-1 CTLs but not wild-type naïve CD8⁺ T cells established functional GJs with B16F10 cells. Interestingly, we observed that Cx43-GJs were required for an efficient granzyme B activity in target B16F10 cells. Using an HLA-A2-restricted/MART-1-specific CD8⁺ T-cell clone, we confirmed these observations in human cells. Our results suggest that Cx43-channels are relevant components of cytotoxic immunological synapses and potentiate CTL-mediated tumor cell killing. Full article

(This article belongs to the Special Issue Connexin-Based Channels in Inflammatory Processes)

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22 pages, 4073 KiB

Open AccessArticle

Role of a RhoA/ROCK-Dependent Pathway on Renal Connexin43 Regulation in the Angiotensin II-Induced Renal Damage

by Gonzalo I. Gómez, Victoria Velarde and Juan C. Sáez

Int. J. Mol. Sci. 2019, 20(18), 4408; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20184408 - 07 Sep 2019

Cited by 17 | Viewed by 3382

Abstract

In various models of chronic kidney disease, the amount and localization of Cx43 in the nephron is known to increase, but the intracellular pathways that regulate these changes have not been identified. Therefore, we proposed that: “In the model of renal damage induced [...] Read more.

In various models of chronic kidney disease, the amount and localization of Cx43 in the nephron is known to increase, but the intracellular pathways that regulate these changes have not been identified. Therefore, we proposed that: “In the model of renal damage induced by infusion of angiotensin II (AngII), a RhoA/ROCK-dependent pathway, is activated and regulates the abundance of renal Cx43”. In rats, we evaluated: 1) the time-point where the renal damage induced by AngII is no longer reversible; and 2) the involvement of a RhoA/ROCK-dependent pathway and its relationship with the amount of Cx43 in this irreversible stage. Systolic blood pressure (SBP) and renal function (urinary protein/urinary creatinine: Uprot/UCrea) were evaluated as systemic and organ outcomes, respectively. In kidney tissue, we also evaluated: 1) oxidative stress (amount of thiobarbituric acid reactive species), 2) inflammation (immunoperoxidase detection of the inflammatory markers ED-1 and IL-1β), 3) fibrosis (immune detection of type III collagen; Col III) and 4) activity of RhoA/ROCK (amount of phosphorylated MYPT1; p-MYPT1). The ratio Uprot/UCrea, SBP, oxidative stress, inflammation, amount of Cx43 and p-MYPT1 remained high 2 weeks after suspending AngII treatment in rats treated for 4 weeks with AngII. These responses were not observed in rats treated with AngII for less than 4 weeks, in which all measurements returned spontaneously close to the control values after suspending AngII treatment. Rats treated with AngII for 6 weeks and co-treated for the last 4 weeks with Fasudil, an inhibitor of ROCK, showed high SBP but did not present renal damage or increased amount of renal Cx43. Therefore, renal damage induced by AngII correlates with the activation of RhoA/ROCK and the increase in Cx43 amounts and can be prevented by inhibitors of this pathway. Full article

(This article belongs to the Special Issue Connexin-Based Channels in Inflammatory Processes)

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17 pages, 7547 KiB

Open AccessArticle

Gadolinium Chloride Restores the Function of the Gap Junctional Intercellular Communication between Hepatocytes in a Liver Injury

by Le Yang, Chengbin Dong, Lei Tian, Xiaofang Ji, Lin Yang and Liying Li

Int. J. Mol. Sci. 2019, 20(15), 3748; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20153748 - 31 Jul 2019

Cited by 7 | Viewed by 2575

Abstract

Background: Gadolinium chloride (GdCl3) has been reported to attenuate liver injury caused by a variety of toxicants. Gap junctional intercellular communication (GJIC) is thought to be essential in controlling liver homeostasis and pathology. Here we evaluate the effects of GdCl3 on functional GJIC [...] Read more.

Background: Gadolinium chloride (GdCl3) has been reported to attenuate liver injury caused by a variety of toxicants. Gap junctional intercellular communication (GJIC) is thought to be essential in controlling liver homeostasis and pathology. Here we evaluate the effects of GdCl3 on functional GJIC and connexin expression in mouse models and primary hepatocytes. Methods: Mice were administered GdCl3 intraperitoneally the day before a carbon tetrachloride (CCl4) injection or bile duct ligation (BDL) operation. Primary hepatocytes were treated with CCl4 or lipopolysaccharides (LPS), with or without GdCl3. A scrape loading/dye transfer assay was performed to assess the GJIC function. The expression of connexins was examined by real-time reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescent staining. Results: CCl4 treatment or the BDL operation led to the dysfunction of GJIC and a down-regulation of Cx32 and Cx26 in injured liver. GdCl3 administration restored GJIC function between hepatocytes by facilitating the transfer of fluorescent dye from one cell into adjacent cells via GJIC, and markedly prevented the decrease of Cx32 and Cx26 in injured liver. In primary hepatocytes, CCl4 or LPS treatment induced an obvious decline of Cx32 and Cx26, whereas GdCl3 pretreatment prevented the down-regulation of connexins. In vivo GdCl3 protected hepatocytes and attenuated the liver inflammation and fibrosis in liver injury mouse models. Conclusion: GdCl3 administration protects functional GJIC between hepatocytes, and prevents the decrease of connexin proteins at mRNA and protein levels during liver injury, leading to the alleviation of chronic liver injury. Full article

(This article belongs to the Special Issue Connexin-Based Channels in Inflammatory Processes)

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Review

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18 pages, 1656 KiB

Open AccessReview

Role of ROS/RNS in Preeclampsia: Are Connexins the Missing Piece?

by María F. Rozas-Villanueva, Paola Casanello and Mauricio A. Retamal

Int. J. Mol. Sci. 2020, 21(13), 4698; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21134698 - 30 Jun 2020

Cited by 11 | Viewed by 3918

Abstract

Preeclampsia is a pregnancy complication that appears after 20 weeks of gestation and is characterized by hypertension and proteinuria, affecting both mother and offspring. The cellular and molecular mechanisms that cause the development of preeclampsia are poorly understood. An important feature of preeclampsia [...] Read more.

Preeclampsia is a pregnancy complication that appears after 20 weeks of gestation and is characterized by hypertension and proteinuria, affecting both mother and offspring. The cellular and molecular mechanisms that cause the development of preeclampsia are poorly understood. An important feature of preeclampsia is an increase in oxygen and nitrogen derived free radicals (reactive oxygen species/reactive nitrogen species (ROS/RNS), which seem to be central players setting the development and progression of preeclampsia. Cell-to-cell communication may be disrupted as well. Connexins (Cxs), a family of transmembrane proteins that form hemichannels and gap junction channels (GJCs), are essential in paracrine and autocrine cell communication, allowing the movement of signaling molecules between cells as well as between the cytoplasm and the extracellular media. GJCs and hemichannels are fundamental for communication between endothelial and smooth muscle cells and, therefore, in the control of vascular contraction and relaxation. In systemic vasculature, the activity of GJCs and hemichannels is modulated by ROS and RNS. Cxs participate in the development of the placenta and are expressed in placental vasculature. However, it is unknown whether Cxs are modulated by ROS/RNS in the placenta, or whether this potential modulation contributes to the pathogenesis of preeclampsia. Our review addresses the possible role of Cxs in preeclampsia, and the plausible modulation of Cxs-formed channels by ROS and RNS. We suggest these factors may contribute to the development of preeclampsia. Full article

(This article belongs to the Special Issue Connexin-Based Channels in Inflammatory Processes)

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26 pages, 2406 KiB

Open AccessReview

Gap Junctions in the Bone Marrow Lympho-Hematopoietic Stem Cell Niche, Leukemia Progression, and Chemoresistance

by Abhishek K. Singh and Jose A. Cancelas

Int. J. Mol. Sci. 2020, 21(3), 796; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21030796 - 25 Jan 2020

Cited by 24 | Viewed by 9451

Abstract

The crosstalk between hematopoietic stem cells (HSC) and bone marrow (BM) microenvironment is critical for homeostasis and hematopoietic regeneration in response to blood formation emergencies after injury, and has been associated with leukemia transformation and progression. Intercellular signals by the BM stromal cells [...] Read more.

The crosstalk between hematopoietic stem cells (HSC) and bone marrow (BM) microenvironment is critical for homeostasis and hematopoietic regeneration in response to blood formation emergencies after injury, and has been associated with leukemia transformation and progression. Intercellular signals by the BM stromal cells in the form of cell-bound or secreted factors, or by physical interaction, regulate HSC localization, maintenance, and differentiation within increasingly defined BM HSC niches. Gap junctions (GJ) are comprised of arrays of membrane embedded channels formed by connexin proteins, and control crucial signaling functions, including the transfer of ions, small metabolites, and organelles to adjacent cells which affect intracellular mechanisms of signaling and autophagy. This review will discuss the role of GJ in both normal and leukemic hematopoiesis, and highlight some of the most novel approaches that may improve the efficacy of cytotoxic drugs. Connexin GJ channels exert both cell-intrinsic and cell-extrinsic effects on HSC and BM stromal cells, involved in regenerative hematopoiesis after myelosuppression, and represent an alternative system of cell communication through a combination of electrical and metabolic coupling as well as organelle transfer in the HSC niche. GJ intercellular communication (GJIC) in the HSC niche improves cellular bioenergetics, and rejuvenates damaged recipient cells. Unfortunately, they can also support leukemia proliferation and survival by creating leukemic niches that provide GJIC dependent energy sources and facilitate chemoresistance and relapse. The emergence of new strategies to disrupt self-reinforcing malignant niches and intercellular organelle exchange in leukemic niches, while at the same time conserving normal hematopoietic GJIC function, could synergize the effect of chemotherapy drugs in eradicating minimal residual disease. An improved understanding of the molecular basis of connexin regulation in normal and leukemic hematopoiesis is warranted for the re-establishment of normal hematopoiesis after chemotherapy. Full article

(This article belongs to the Special Issue Connexin-Based Channels in Inflammatory Processes)

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15 pages, 650 KiB

Open AccessReview

Connexin 43 Mutations Lead to Increased Hemichannel Functionality in Skin Disease

by Anthony G. Cocozzelli and Thomas W. White

Int. J. Mol. Sci. 2019, 20(24), 6186; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20246186 - 07 Dec 2019

Cited by 24 | Viewed by 3564

Abstract

Gap junctional channels are specialized components of the cellular membrane that allow the intercellular passage of small metabolites, ions, and second messengers to maintain homeostasis. They are comprised of members of the connexin gene family that encode a wide array of proteins that [...] Read more.

Gap junctional channels are specialized components of the cellular membrane that allow the intercellular passage of small metabolites, ions, and second messengers to maintain homeostasis. They are comprised of members of the connexin gene family that encode a wide array of proteins that are expressed in nearly every tissue type. Cx43 is perceived to be the most broadly expressed connexin in humans, with several genetic skin diseases being linked to Cx43 mutations specifically. These mutations, in large, produce a gain of functional hemichannels that contribute to the phenotypes of Erythrokeratoderma Variabilis et Progressiva (EKVP), Palmoplantar Keratodemra Congenital Alopecia-1 (PPKCA1), and others that produce large conductance and increased permselectivity in otherwise quiescent structures. Gaining functional hemichannels can have adverse effects in the skin, inducing apoptosis via Ca²⁺ overload or increased ATP permeability. Here, we review the link between Cx43 and skin disease. We aim to provide insight into the mechanisms regulating the normal and pathophysiological gating of these essential proteins, as well as address current therapeutic strategies. We also demonstrate that transient transfection of neuro-2a (N2a) cells with mutant Cx43 cDNA resulted in increased hemichannel activity compared to wild-type Cx43 and untransfected cells, which is consistent with other studies in the current literature. Full article

(This article belongs to the Special Issue Connexin-Based Channels in Inflammatory Processes)

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17 pages, 537 KiB

Open AccessReview

Connexins-Based Hemichannels/Channels and Their Relationship with Inflammation, Seizures and Epilepsy

by Laura Medina-Ceja, Juan C. Salazar-Sánchez, Jorge Ortega-Ibarra and Alberto Morales-Villagrán

Int. J. Mol. Sci. 2019, 20(23), 5976; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20235976 - 27 Nov 2019

Cited by 29 | Viewed by 4381

Abstract

Connexins (Cxs) are a family of 21 protein isoforms, eleven of which are expressed in the central nervous system, and they are found in neurons and glia. Cxs form hemichannels (connexons) and channels (gap junctions/electric synapses) that permit functional and metabolic coupling between [...] Read more.

Connexins (Cxs) are a family of 21 protein isoforms, eleven of which are expressed in the central nervous system, and they are found in neurons and glia. Cxs form hemichannels (connexons) and channels (gap junctions/electric synapses) that permit functional and metabolic coupling between neurons and astrocytes. Altered Cx expression and function is involved in inflammation and neurological diseases. Cxs-based hemichannels and channels have a relevance to seizures and epilepsy in two ways: First, this pathological condition increases the opening probability of hemichannels in glial cells to enable gliotransmitter release, sustaining the inflammatory process and exacerbating seizure generation and epileptogenesis, and second, the opening of channels favors excitability and synchronization through coupled neurons. These biological events highlight the global pathological mechanism of epilepsy, and the therapeutic potential of Cxs-based hemichannels and channels. Therefore, this review describes the role of Cxs in neuroinflammation and epilepsy and examines how the blocking of channels and hemichannels may be therapeutic targets of anti-convulsive and anti-epileptic treatments. Full article

(This article belongs to the Special Issue Connexin-Based Channels in Inflammatory Processes)

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