Connexin26 is regulated in rat urothelium by the scaffold protein IB1/JIP-1.

Proper function of the wall of bladder requires gap junctional communication for coordinating the responses of smooth muscle (SMC) and urothelial cells exposed to urine pressure. In the rat bladder, Cx43 is expressed by SMC and urothelial cells, whereas Cx26 expression is restricted to the epithelium. We used a model of bladder outlet obstruction, in which a ligature is placed around the urethra to increase voiding pressure. Increased fluid pressure was associated with increased Cx43 and Cx26 mRNA expression and with the activation of a signaling cascade including the transcription factor c-Jun, which is a component of the AP-1 complex. The signaling pathway of the c-Jun NH2 terminal kinase (JNK) requires the presence of the scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1). Under stress conditions resulting from urine retention, we have found a reduced content of IB1/JIP-1 in urothelial cells, which in turn induced a drastic increase of JNK and AP-1 binding activities. The stress-induced activation of JNK was prevented by overexpressing IB1/JIP-1, using a viral gene transfer approach, a condition which also resulted in a decrease in Cx26 mRNA. The data show that: 1) mechanical stress of urothelial cells activates in vivo JNK, as a consequence of a regulated expression of IB1/JIP-1 and 2) that urothelial Cx26 may be directly regulated by the AP-1 complex.

Increased connexin43 expression in human saphenous veins in culture is associated with intimal hyperplasia.

OBJECTIVE: Intimal hyperplasia is a vascular remodelling process that occurs after a vascular injury. The mechanisms involved in intimal hyperplasia are proliferation, dedifferentiation, and migration of medial smooth muscle cells towards the subintimal space. We postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, might participate in the development of intimal hyperplasia. Connexin43 (Cx43) expression levels may be altered in intimal hyperplasia, and we therefore evaluated the regulated expression of Cx43 in human saphenous veins in culture in the presence or not of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. METHODS: Segments of harvested human saphenous veins, obtained at the time of bypass graft, were opened longitudinally with the luminal surface uppermost and maintained in culture for 14 days. Vein fragments were then processed for histologic examination, neointimal thickness measurements, immunocytochemistry, RNA, and proteins analysis. RESULTS: Of the four connexins (Cx37, 40, 43, and 45), we focused on Cx43 and Cx40, which we found by real-time polymerase chain reaction to be expressed in the saphenous vein because they are the predominant connexins expressed by smooth muscle cells and endothelial cells. After 14 days of culture, histomorphometric analysis showed a significant increase in the intimal thickness as observed during the process of intimal hyperplasia. A time-course analysis revealed a progressive upregulation of Cx43 to reach a maximal increase of sixfold to eightfold at both transcript and protein levels after 14 days in culture. In contrast, the expression of Cx40, abundantly expressed in the endothelial cells, was not altered. Immunofluorescence showed a large increase in Cx43 within smooth muscle cell membranes of the media layer. The development of intimal hyperplasia in vitro was decreased in presence of fluvastatin and was associated with reduced Cx43 expression. CONCLUSIONS: These data show that Cx43 is increased in vitro during the process of intimal hyperplasia and that fluvastatin could prevent this induction, supporting a critical role for Cx43-mediated gap-junctional communication in the human vein during the development of intimal hyperplasia. CLINICAL RELEVANCE: Stenosis due to intimal hyperplasia is the most common cause of failure of venous bypass grafts. To better understand the development of intimal hyperplasia, we used an ex vivo organ culture model to study saphenous veins harvested from patients undergoing a lower limb bypass surgery. In this model, the morphologic and functional integrity of the vessel wall is maintained and significant intimal hyperplasia development occurs after 14 days in culture. We have postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, may participate in the development of intimal hyperplasia. Indeed, intimal hyperplasia consists of proliferation and migration of smooth muscle cells into the subendothelial space. Intercellular communication is responsible for the direct transfer of ions and small molecules from one cell to the other through gap-junction channels found at cell-cell appositions. No study to date has evaluated whether gap junctional communication is involved in the process of intimal hyperplasia in humans. This assertion was investigated by using the aforementioned organ culture model of intimal hyperplasia in human saphenous veins, and our data support a critical role for Cx43-mediated gap junctional communication in human vein during the development of intimal hyperplasia.

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43.

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.

Control of the proliferation of activated CD4+ T cells by connexins.

As expression of Cxs in cells of the immune system increases upon cellular activation, we investigated whether Cxs and especially CxHcs play a major role during T cell-mediated responses. In particular, we studied the expression of Cx43Hc following CD4(+) T cell stimulation using flow cytometry, real-time PCR, and Western blot analysis. We showed that expression of Cx43 and its phosphorylated isoforms increased in response to the engagement of CD3 and CD28. Cx43Hcs were found to be involved in sustaining proliferation of T cells, as assessed by cell cycle staining, thymidine incorporation assays, and CFSE analysis of cells exposed to mimetic peptide inhibitors of the plasma membrane Cx channels and antibodies generated to an extracellular region of Cx. The reduction of T cell proliferation mediated by Cx channel inhibitors suppressed cysteine uptake but not cytokine production. We conclude that upon antigen recognition, T cells require CxHc to sustain their clonal expansion.

Connexins protect mouse pancreatic β cells against apoptosis.

Type 1 diabetes develops when most insulin-producing β cells of the pancreas are killed by an autoimmune attack. The in vivo conditions modulating the sensitivity and resistance of β cells to this attack remain largely obscure. Here, we show that connexin 36 (Cx36), a trans-membrane protein that forms gap junctions between β cells in the pancreatic islets, protects mouse β cells against both cytotoxic drugs and cytokines that prevail in the islet environment at the onset of type 1 diabetes. We documented that this protection was at least partially dependent on intercellular communication, which Cx36 and other types of connexin channels establish within pancreatic islets. We further found that proinflammatory cytokines decreased expression of Cx36 and that experimental reduction or augmentation of Cx36 levels increased or decreased β cell apoptosis, respectively. Thus, we conclude that Cx36 is central to β cell protection from toxic insults.

Connexins: key mediators of endocrine function.

The appearance of multicellular organisms imposed the development of several mechanisms for cell-to-cell communication, whereby different types of cells coordinate their function. Some of these mechanisms depend on the intercellular diffusion of signal molecules in the extracellular spaces, whereas others require cell-to-cell contact. Among the latter mechanisms, those provided by the proteins of the connexin family are widespread in most tissues. Connexin signaling is achieved via direct exchanges of cytosolic molecules between adjacent cells at gap junctions, for cell-to-cell coupling, and possibly also involves the formation of membrane "hemi-channels," for the extracellular release of cytosolic signals, direct interactions between connexins and other cell proteins, and coordinated influence on the expression of multiple genes. Connexin signaling appears to be an obligatory attribute of all multicellular exocrine and endocrine glands. Specifically, the experimental evidence we review here points to a direct participation of the Cx36 isoform in the function of the insulin-producing β-cells of the endocrine pancreas, and of the Cx40 isoform in the function of the renin-producing juxtaglomerular epithelioid cells of the kidney cortex.

Connexin 43 expression in human hypertrophied heart due to pressure and volume overload.

Left ventricular hypertrophy (LVH) is due to pressure overload or mechanical stretch and is thought to be associated with remodeling of gap-junctions. We investigated whether the expression of connexin 43 (Cx43) is altered in humans in response to different degrees of LVH. The expression of Cx43 was analyzed by quantitative polymerase chain reaction, Western blot analysis and immunohistochemistry on left ventricular biopsies from patients undergoing aortic or mitral valve replacement. Three groups were analyzed: patients with aortic stenosis with severe LVH (n=9) versus only mild LVH (n=7), and patients with LVH caused by mitral regurgitation (n=5). Cx43 mRNA expression and protein expression were similar in the three groups studied. Furthermore, immunohistochemistry revealed no change in Cx43 distribution. We can conclude that when compared with mild LVH or with LVH due to volume overload, severe LVH due to chronic pressure overload is not accompanied by detectable changes of Cx43 expression or spatial distribution.

Intercellular communication in atherosclerosis.

Cell-to-cell communication is a process necessary for physiological tissue homeostasis and appears often altered during disease. Gap junction channels, formed by connexins, allow the direct intercellular communication between adjacent cells. After a brief review of the pathophysiology of atherosclerosis, we will discuss the role of connexins throughout the different stages of the disease.

Fast oscillatory activity in the anterior cingulate cortex: dopaminergic modulation and effect of perineuronal net loss.

Dopamine release in the prefrontal cortex plays a critical role in cognitive function such as working memory, attention and planning. Dopamine exerts complex modulation on excitability of pyramidal neurons and interneurons, and regulates excitatory and inhibitory synaptic transmission. Because of the complexity of this modulation, it is difficult to fully comprehend the effect of dopamine on neuronal network activity. In this study, we investigated the effect of dopamine on local high-frequency oscillatory neuronal activity (in β band) in slices of the mouse anterior cingulate cortex (ACC). We found that dopamine enhanced the power of these oscillations induced by kainate and carbachol, but did not affect their peak frequency. Activation of D2R and in a lesser degree D1R increased the oscillation power, while activation of D4R had no effect. These high-frequency oscillations in the ACC relied on both phasic inhibitory and excitatory transmission and functional gap junctions. Thus, dopamine released in the ACC promotes high-frequency synchronized local cortical activity which is known to favor information transfer, fast selection and binding of distributed neuronal responses. Finally, the power of these oscillations was significantly enhanced after degradation of the perineuronal nets (PNNs) enwrapping most parvalbumin interneurons. This study provides new insights for a better understanding of the abnormal prefrontal gamma activity in schizophrenia (SZ) patients who display prefrontal anomalies of both the dopaminergic system and the PNNs.

Expression and distribution of connexin 32 in rat liver with experimentally induced fibrosis

The connexin 32 (Cx32) is a protein that forms the channels that promote the gap junction intercellular communication (GJIC) in the liver, allowing the diffusion of small molecules through cytosol from cell-to-cell. Hepatic fibrosis is characterized by a disruption of normal tissue architeture by cellular lesions, and may alter the GJIC. This work aimed to study the expression and distribution of Cx32 in liver fibrosis induced by the oral administration of dimethylnitrosamine in female Wistar rats. The necropsy of the rats was carried out after five weeks of drug administration. They presented a hepatic fibrosis state. Sections from livers with fibrosis and from control livers were submitted to immunohistochemical, Real Time-PCR and Western-Blot analysis to Cx32. In fibrotic livers the Cxs were diffusely scattered in the cytoplasm, contrasting with the control livers, where the Cx32 formed junction plaques at the cell membrane. Also it was found a decrease in the gene expression of Cx32 without reduction in the protein quantity when compared with controls. These results suggest that there the mechanism of intercellular communication between hepatocytes was reduced by the fibrotic process, which may predispose to the occurrence of a neoplastic process, taken in account that connexins are considered tumor suppressing genes.

A simple RT-PCR-based strategy for screening connexin identity

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

The molecular basis of selective permeability of connexins is complex and includes both size and charge

Although gap junction channels are still widely viewed as large, non-specific pores connecting cells, the diversity in the connexin family has led more attention to be focused on their permeability characteristics. We summarize here the current status of these investigations, both published and on-going, that reveal both charge and size selectivity between gap junction channels composed of different connexins. In particular, this review will focus on quantitative approaches that monitor the expression level of the connexins, so that it is clear that differences that are seen can be attributed to channel properties. The degree of selectivity that is observed is modest compared to other channels, but is likely to be significant for biological molecules that are labile within the cell. Of particular relevance to the in vivo function of gap junctions, recent studies are summarized that demonstrate that the connexin phenotype can control the nature of the endogenous traffic between cells, with consequent effects on biological effects of gap junctions such as tumor suppression.

Regulation of intercellular coupling in acute and chronic heart disease

Effective pump function of the heart depends on the precise control of spatial and temporal patterns of electrical activation. Accordingly, the distribution and function of gap junction channels are important determinants of the conduction properties of myocardium and undoubtedly play other roles in intercellular communication crucial to normal cardiac function. Recent advances have begun to elucidate mechanisms by which the heart regulates intercellular electrical coupling at gap junctions in response to stress or injury. Although responses to increased load or injury are generally adaptive in nature, remodeling of intercellular junctions under conditions of severe stress creates anatomic substrates conducive to the development of lethal ventricular arrhythmias. Potential mechanisms controlling the level of intercellular communication in the heart include regulation of connexin turnover dynamics and phosphorylation.

Chronic hypertension alters the expression of Cx43 in cardiovascular muscle cells

Connexin43 (Cx43), the predominant gap junction protein of muscle cells in vessels and heart, is involved in the control of cell-to-cell communication and is thought to modulate the contractility of the vascular wall and the electrical coupling of cardiac myocytes. We have investigated the effects of arterial hypertension on the expression of Cx43 in aorta and heart in three different models of experimental hypertension. Rats were made hypertensive either by clipping one renal artery (two kidney, one-clip renal (2K,1C) model) by administration of deoxycorticosterone and salt (DOCA-salt model) or by inhibiting nitric oxide synthase with NG-nitro-L-arginine methyl ester (L-NAME model). After 4 weeks, rats of the three models showed a similar increase in intra-arterial mean blood pressure and in the thickness of the walls of both aorta and heart. Analysis of heart mRNA demonstrated no change in Cx43 expression in the three models compared to their respective controls. The same 2K,1C and DOCA-salt hypertensive animals expressed twice more Cx43 in aorta, and the 2K,1C rats showed an increase in arterial distensibility. In contrast, the aortae of L-NAME hypertensive rats were characterized by a 50% decrease in Cx43 and the carotid arteries did not show increased distensibility. Western blot analysis indicated that Cx43 was more phosphorylated in the aortae of 2K,1C rats than in those of L-NAME or control rats, indicating a differential regulation of aortic Cx43 in different models of hypertension. The data suggest that localized mechanical forces induced by hypertension affect Cx43 expression and that the cell-to-cell communication mediated by Cx43 channels may contribute to regulating the elasticity of the vascular wall.

Cell migration in the postnatal subventricular zone

New neurons are constantly added to the olfactory bulb of rodents from birth to adulthood. This accretion is not only dependent on sustained neurogenesis, but also on the migration of neuroblasts and immature neurons from the cortical and striatal subventricular zone (SVZ) to the olfactory bulb. Migration along this long tangential pathway, known as the rostral migratory stream (RMS), is in many ways opposite to the classical radial migration of immature neurons: it is faster, spans a longer distance, does not require radial glial guidance, and is not limited to postmitotic neurons. In recent years many molecules have been found to be expressed specifically in this pathway and to directly affect this migration. Soluble factors with inhibitory, attractive and inductive roles in migration have been described, as well as molecules mediating cell-to-cell and cell-substrate interactions. However, it is still unclear how the various molecules and cells interact to account for the special migratory behavior in the RMS. Here we will propose some candidate mechanisms for roles in initiating and stopping SVZ/RMS migration.