Pablo J. Sáez

Cx43 Hemichannels and Gap Junction Channels in Astrocytes Are Regulated Oppositely by Proinflammatory Cytokines Released from Activated Microglia

Astrocytes have a role in maintaining normal neuronal functions, some of which depend on connexins, protein subunits of gap junction channels and hemichannels. Under inflammatory conditions, microglia release cytokines, including interleukin-1β and tumor necrosis factor-α, that reduce intercellular communication via gap junctions. Now, we demonstrate that either conditioned medium harvested from activated microglia or a mixture of these two cytokines enhances the cellular exchange with the extracellular milieu via Cx43 hemichannels. These changes in membrane permeability were not detected in astrocytes cultured from Cx43 knock-out mice and were abrogated by connexin hemichannel blockers, including La3+, mimetic peptides, and niflumic acid. Both the reduction in gap junctional communication and the increase in membrane permeability were mediated by a p38 mitogen-activated protein kinase-dependent pathway. However, the increase in membrane permeability, but not the gap junction inhibition, was rapidly reversed by the sulfhydryl reducing agent dithiothreitol, indicating that final regulatory mechanisms are different. Treatment with proinflammatory cytokines reduced the total and cell surface Cx43 levels, suggesting that the increase in membrane permeability was attributable to an increase in hemichannels activity. Indeed, unitary events of ∼220 pS corresponding to Cx43 hemichannels were much more frequent in astrocytes treated with microglia conditioned medium than under control conditions. Finally, the effect of cytokines enhanced the uptake and reduced the intercellular diffusion of glucose, which might explain changes in the metabolic status of astrocytes under inflammatory conditions. Accordingly, this opposite regulation may affect glucose trafficking and certainly will modify the metabolic status of astrocytes involved in brain inflammation.

Modulation of Brain Hemichannels and Gap Junction Channels by Pro-Inflammatory Agents and Their Possible Role in Neurodegeneration

In normal brain, neurons, astrocytes, and oligodendrocytes, the most abundant and active cells express pannexins and connexins, protein subunits of two families forming membrane channels. Most available evidence indicates that in mammals endogenously expressed pannexins form only hemichannels and connexins form both gap junction channels and hemichannels. Whereas gap junction channels connect the cytoplasm of contacting cells and coordinate electric and metabolic activity, hemichannels communicate the intra- and extracellular compartments and serve as a diffusional pathway for ions and small molecules. A subthreshold stimulation by acute pathological threatening conditions (e.g., global ischemia subthreshold for cell death) enhances neuronal Cx36 and glial Cx43 hemichannel activity, favoring ATP release and generation of preconditioning. If the stimulus is sufficiently deleterious, microglia become overactivated and release bioactive molecules that increase the activity of hemichannels and reduce gap junctional communication in astroglial networks, depriving neurons of astrocytic protective functions, and further reducing neuronal viability. Continuous glial activation triggered by low levels of anomalous proteins expressed in several neurodegenerative diseases induce glial hemichannel and gap junction channel disorders similar to those of acute inflammatory responses triggered by ischemia or infectious diseases. These changes are likely to occur in diverse cell types of the CNS and contribute to neurodegeneration during inflammatory process.

Amyloid β-Induced Death in Neurons Involves Glial and Neuronal Hemichannels

The mechanisms involved in Alzheimers disease are not completely understood and how glial cells contribute to this neurodegenerative disease remains to be elucidated. Because inflammatory treatments and products released from activated microglia increase glial hemichannel activity, we investigated whether amyloid-β peptide (Aβ) could regulate these channels in glial cells and affect neuronal viability. Microglia, astrocytes, or neuronal cultures as well as acute hippocampal slices made from GFAP-eGFP transgenic mice were treated with the active fragment of Aβ. Hemichannel activity was monitored by single-channel recordings and by time-lapse ethidium uptake, whereas neuronal death was assessed by Fluoro-Jade C staining. We report that low concentrations of Aβ25–35 increased hemichannel activity in all three cell types and microglia initiate these effects triggered by Aβ. Finally, neuronal damage occurs by activation of neuronal hemichannels induced by ATP and glutamate released from Aβ25–35-activated glia. These responses were observed in the presence of external calcium and were differently inhibited by hemichannel blockers, whereas the Aβ25–35-induced neuronal damage was importantly reduced in acute slices made from Cx43 knock-out mice. Thus, Aβ leads to a cascade of hemichannel activation in which microglia promote the release of glutamate and ATP through glial (microglia and astrocytes) hemichannels that induces neuronal death by triggering hemichannels in neurons. Consequently, this work opens novel avenues for alternative treatments that target glial cells and neurons to maintain neuronal survival in the presence of Aβ.

Glucose increases intracellular free Ca2+ in tanycytes via ATP released through connexin 43 hemichannels

The ventromedial hypothalamus is involved in regulating feeding and satiety behavior, and its neurons interact with specialized ependymal‐glial cells, termed tanycytes. The latter express glucose‐sensing proteins, including glucose transporter 2, glucokinase, and ATP‐sensitive K+ (KATP) channels, suggesting their involvement in hypothalamic glucosensing. Here, the transduction mechanism involved in the glucose‐induced rise of intracellular free Ca2+ concentration ([Ca2+]i) in cultured β‐tanycytes was examined. Fura‐2AM time‐lapse fluorescence images revealed that glucose increases the intracellular Ca2+ signal in a concentration‐dependent manner. Glucose transportation, primarily via glucose transporters, and metabolism via anaerobic glycolysis increased connexin 43 (Cx43) hemichannel activity, evaluated by ethidium uptake and whole cell patch clamp recordings, through a KATP channel‐dependent pathway. Consequently, ATP export to the extracellular milieu was enhanced, resulting in activation of purinergic P2Y1 receptors followed by inositol trisphosphate receptor activation and Ca2+ release from intracellular stores. The present study identifies the mechanism by which glucose increases [Ca2+]i in tanycytes. It also establishes that Cx43 hemichannels can be rapidly activated under physiological conditions by the sequential activation of glucosensing proteins in normal tanycytes.

Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells

Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA–mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42–Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4–MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.

Mesenchymal stem cell injection ameliorates chronic renal failure in a rat model.

CKD (chronic kidney disease) has become a public health problem. The therapeutic approaches have been able to reduce proteinuria, but have not been successful in limiting disease progression. In this setting, cell therapies associated with regenerative effects are attracting increasing interest. We evaluated the effect of MSC (mesenchymal stem cells) on the progression of CKD and the expression of molecular biomarkers associated with regenerative effects. Adult male Sprague-Dawley rats subjected to 5/6 NPX (nephrectomy) received a single intravenous infusion of 0.5×106 MSC or culture medium. A sham group subjected to the same injection was used as the control. Rats were killed 5 weeks after MSC infusion. Dye tracking of MSC was followed by immunofluorescence analysis. Kidney function was evaluated using plasma creatinine. Structural damage was evaluated by H&E (haematoxylin and eosin) staining, ED-1 abundance (macrophages) and interstitial α-SMA (α-smooth muscle actin). Repairing processes were evaluated by functional and structural analyses and angiogenic/epitheliogenic protein expression. MSC could be detected in kidney tissues from NPX animals treated with intravenous cell infusion. This group presented a marked reduction in plasma creatinine levels and damage markers ED-1 and α-SMA (P<0.05). In addition, treated rats exhibited a significant induction in epitheliogenic [Pax-2, bFGF (basic fibroblast growth factor) and BMP-7 (bone morphogenetic protein-7)] and angiogenic [VEGF (vascular endothelial growth factor) and Tie-2] proteins. The expression of these biomarkers of regeneration was significantly related to the increase in renal function. Many aspects of the cell therapy in CKD remain to be investigated in more detail: for example, its safety, low cost and the possible need for repeated cell injections over time. Beyond the undeniable importance of these issues, what still needs to be clarified is whether MSC administration has a real effect on the treatment of this pathology. It is precisely to this point that the present study aims to contribute.

Human mesenchymal stem cells derived from adipose tissue reduce functional and tissue damage in a rat model of chronic renal failure

Therapeutic approaches for CKD (chronic kidney disease) have been able to reduce proteinuria, but not diminish the disease progression. We have demonstrated beneficial effects by injection of BM (bone marrow)-derived MSCs (mesenchymal stem cells) from healthy donors in a rat model with CKD. However, it has recently been reported that BM-MSCs derived from uraemic patients failed to confer functional protection in a similar model. This suggests that autologous BM-MSCs are not suitable for the treatment of CKD. In the present study, we have explored the potential of MSCs derived from adipose tissue (AD-MSCs) as an alternative source of MSCs for the treatment of CKD. We have isolated AD-MSCs and evaluated their effect on the progression of CKD. Adult male SD (Sprague-Dawley) rats subjected to 5/6 NPX (nephrectomy) received a single intravenous infusion of 0.5×10(6) AD-MSCs or MSC culture medium alone. The therapeutic effect was evaluated by plasma creatinine measurement, structural analysis and angiogenic/epitheliogenic protein expression. AD-MSCs were detected in kidney tissues from NPX animals. This group had a significant reduction in plasma creatinine levels and a lower expression of damage markers ED-1 and α-SMA (α-smooth muscle actin) (P<0.05). In addition, treated rats exhibited a higher level of epitheliogenic [Pax-2 and BMP-7 (bone morphogenetic protein 7)] and angiogenic [VEGF (vascular endothelial growth factor)] proteins. The expression of these biomarkers of regeneration was significantly related to the improvement in renal function. Although many aspects of the cell therapy for CKD remain to be investigated, we provide evidence that AD-MSCs, a less invasive and highly available source of MSCs, exert an important therapeutic effect in this pathology.

Regulation of Hemichannels and Gap Junction Channels by Cytokines in Antigen-Presenting Cells

Autocrine and paracrine signals coordinate responses of several cell types of the immune system that provide efficient protection against different challenges. Antigen-presenting cells (APCs) coordinate activation of this system via homocellular and heterocellular interactions. Cytokines constitute chemical intercellular signals among immune cells and might promote pro- or anti-inflammatory effects. During the last two decades, two membrane pathways for intercellular communication have been demonstrated in cells of the immune system. They are called hemichannels (HCs) and gap junction channels (GJCs) and provide new insights into the mechanisms of the orchestrated response of immune cells. GJCs and HCs are permeable to ions and small molecules, including signaling molecules. The direct intercellular transfer between contacting cells can be mediated by GJCs, whereas the release to or uptake from the extracellular milieu can be mediated by HCs. GJCs and HCs can be constituted by two protein families: connexins (Cxs) or pannexins (Panxs), which are present in almost all APCs, being Cx43 and Panx1 the most ubiquitous members of each protein family. In this review, we focus on the effects of different cytokines on the intercellular communication mediated by HCs and GJCs in APCs and their impact on purinergic signaling.

ATP Is Required and Advances Cytokine-Induced Gap Junction Formation in Microglia In Vitro

Microglia are the immune cells in the central nervous system. After injury microglia release bioactive molecules, including cytokines and ATP, which modify the functional state of hemichannels (HCs) and gap junction channels (GJCs), affecting the intercellular communication via extracellular and intracellular compartments, respectively. Here, we studied the role of extracellular ATP and several cytokines as modulators of the functional state of microglial HCs and GJCs using dye uptake and dye coupling techniques, respectively. In microglia and the microglia cell line EOC20, ATP advanced the TNF-α/IFN-γ-induced dye coupling, probably through the induction of IL-1β release. Moreover, TNF-α/IFN-γ, but not TNF-α plus ATP, increased dye uptake in EOC20 cells. Blockade of Cx43 and Panx1 HCs prevented dye coupling induced by TNF-α/IFN-γ, but not TNF-α plus ATP. In addition, IL-6 prevented the induction of dye coupling and HC activity induced by TNF-α/IFN-γ in EOC20 cells. Our data support the notion that extracellular ATP affects the cellular communication between microglia through autocrine and paracrine mechanisms, which might affect the timing of immune response under neuroinflammatory conditions.

Injury of skeletal muscle and specific cytokines induce the expression of gap junction channels in mouse dendritic cells

Dendritic cells (DCs) in culture express at least connexin43, a protein subunit of gap junctions, and form gap junction channels, which could be important for T‐cells activation. Here, we evaluated whether DCs express connexins in vivo and also to identify components of their microenvironment that regulate the functional expression of gap junctions. In vivo studies were performed in lymph nodes of mice under control conditions or after skeletal muscle damage. In double immunolabeling studies, connexin45 was frequently detected in DEC205+ DCs in lymph nodes of control animals, whereas connexin43 was rarely found in DCs. However, connexin43 was upregulated in DCs after skeletal muscle damage. Upregulation of connexin43 gene expression by tissue damage was also confirmed in mice carrying a β‐galactosidase reporter gene in a connexin43 allele. The effect of several cytokines on the expression of functional gap junctions between cultured DCs was also tested. Under control conditions, cultured DCs did not communicate via gap junctions. However, after treatment with keratinocyte‐conditioned medium or cytokine mixtures containing at least TNF‐α and IL‐1β, they became transiently coupled through a pathway sensitive to octanol, a gap junction blocker. Cellular coupling induced by effective cytokine mixtures was prevented by IL‐6. Single cytokines (TNF‐α, IL‐1β, IFN‐γ, or IL‐6) or other mixtures than the described above did not induce coupling via gap junctions. Increased levels of connexin43 and connexin45 protein and mRNA accompanied the appearance of cellular coupling. These studies provide demonstration of connexin expression and regulation by specific danger signals in DCs. J. Cell. Physiol. 211: 649–660, 2007.