细胞间隙连接通讯与肿瘤

由连接蛋白构成的细胞间隙连接通讯(GJIC)广泛存在于脊椎动物中,可以直接介导细胞间小分子物质的传递,而不需要通过细胞间质。GJIC与肿瘤密切相关,多数肿瘤GJIC能力降低或丧失,连接蛋白不表达或胞内定位,而恢复GJIC可以抑制肿瘤, GJIC已成为肿瘤预防与治疗研究的潜在靶点之一。     

缝隙连接与糖尿病

细胞间通讯方式可分为间接与直接通讯方式,以体循环远程分泌、旁分泌和自分泌方式完成的调节方式为间接通讯,而以细胞间隙连接为途径进行的细胞间直接的信息交流为直接通讯。细胞间隙连接又称缝隙连接,是普遍存在于人和动物组织中的一种细胞连接形式。近年有一系列研究表明缝隙连接胞间通道的异常与糖尿病及其并发症的发生、发展密切相关,本文将就有关这方面的研究进展作一综述。     

Ubiquitination of Gap Junction Proteins

Gap junctions are plasma membrane domains containing arrays of channels that exchange ions and small molecules between neighboring cells. Gap junctional intercellular communication enables cells to directly cooperate both electrically and metabolically. Several lines of evidence indicate that gap junctions are important in regulating cell growth and differentiation and for maintaining tissue homeostasis. Gap junction channels consist of a family of transmembrane proteins called connexins. Gap junctions are dynamic structures, and connexins have a high turnover rate in most tissues. Connexin43 (Cx43), the best-studied connexin isoform, has a half-life of 1.5–5 h; and its degradation involves both the lysosomal and proteasomal systems. Increasing evidence suggests that ubiquitin is important in the regulation of Cx43 endocytosis. Ubiquitination of Cx43 is thought to occur at the plasma membrane and has been shown to be regulated by protein kinase C and the mitogen-activated protein kinase pathway. Cx43 binds to the E3 ubiquitin ligase Nedd4, in a process modulated by Cx43 phosphorylation. The interaction between Nedd4 and Cx43 is mediated by the WW domains of Nedd4 and involves a proline-rich sequence conforming to a PY (XPPXY) consensus motif in the C terminus of Cx43. In addition to the PY motif, an overlapping tyrosine-based sorting signal conforming to the consensus of an YXXϕ motif is involved in Cx43 endocytosis, indicating that endocytosis of gap junctions involves both ubiquitin-dependent and -independent pathways. Here, we discuss current knowledge on the ubiquitination of connexins.     

Gap Junction Channels and Cardiac Impulse Propagation

The role of gap junction channels on cardiac impulse propagation is complex. This review focuses on the differential expression of connexins in the heart and the biophysical properties of gap junction channels under normal and disease conditions. Structural determinants of impulse propagation have been gained from biochemical and immunocytochemical studies performed on tissue extracts and intact cardiac tissue. These have defined the distinctive connexin coexpression patterns and relative levels in different cardiac tissues. Functional determinants of impulse propagation have emerged from electrophysiological experiments carried out on cell pairs. The static properties (channel number and conductance) limit the current flow between adjacent cardiomyocytes and thus set the basic conduction velocity. The dynamic properties (voltage-sensitive gating and kinetics of channels) are responsible for a modulation of the conduction velocity during propagated action potentials. The effect is moderate and depends on the type of Cx and channel. For homomeric-homotypic channels, the influence is small to medium; for homomeric-heterotypic channels, it is medium to strong. Since no data are currently available on heteromeric channels, their influence on impulse propagation is speculative. The modulation by gap junction channels is most prominent in tissues at the boundaries between cardiac tissues such as sinoatrial node-atrial muscle, atrioventricular node-His bundle, His bundle-bundle branch and Purkinje fibers-ventricular muscle. The data predict facilitation of orthodromic propagation.     

Pathophysiological Roles of Gap Junction in Glomerular Mesangial Cells

Glomerular mesangial cells (MCs) are specialized vascular smooth muscle cells that play a critical role in the control of glomerular hemodynamics. One of the intriguing features of MCs is their extraordinary abundance in gap junctions (GJs). It has long been speculated that GJs may bridge MCs together and provide the mesangium with the characteristics of a functional syncytium. Accumulating scientific evidence supports this idea. GJs are reported to be critically involved in important physiological processes like tubuloglomerular feedback and glomerular filtration. In addition, GJs are implicated in the control of many cellular processes of MCs, including growth, differentiation and survival. This article summarizes the current knowledge on the roles of GJs in glomerular pathophysiology.     

Key Connexin 43 Phosphorylation Events Regulate the Gap Junction Life Cycle

Connexin 43 (Cx43), the most widely expressed and abundant vertebrate gap junction protein, is phosphorylated at multiple different serine residues during its life cycle. Cx43 is phosphorylated soon after synthesis and phosphorylation changes as it traffics through the endoplasmic reticulum and Golgi to the plasma membrane, ultimately forming a gap junction structure. The electrophoretic mobility of Cx43 changes as the protein proceeds through its life cycle, with prominent bands often labeled P0, P1 and P2. Many reports have indicated changes in “phosphorylation” based on these mobility shifts and others that occur in response to growth factors or other biological effectors. Here, we indicate how phosphospecific and epitope-specific antibodies can be utilized to show when and where certain phosphorylation events occur during the Cx43 life cycle. These reagents show that phosphorylation at S364 and/or S365 is involved in forming the P1 isoform, an event that apparently regulates trafficking to or within the plasma membrane. Phosphorylation at S325, S328 and/or S330 is necessary to form a P2 isoform; and this phosphorylation event is present only in gap junctions. Treatment with protein kinase C activators led to phosphorylation at S368, S279/S282 and S262 with a shift in mobility in CHO, but not MDCK, cells. The shift was dependent on mitogen-activated protein kinase activity but not phosphorylation at S279/S282. However, phosphorylation at S262 could explain the shift. By defining these phosphorylation events, we have begun to sort out the critical signaling pathways that regulate gap junction function.     

Induction of Nitric Oxide Synthase Inhibits Gap Junction Permeability in Cultured Rat Astrocytes

Abstract: Nitric oxide (^?NO) synthase (NOS) was induced in cultured rat astrocytes by incubation with lipopolysaccharide (LPS) for 18 h and gap junction permeability was assessed by the scrape-loading/Lucifer yellow transfer technique. Induction of NOS was confirmed by determining either the N^G-methyl-l -arginine (NMMA)-inhibitable production of nitrites and nitrates or the conversion of l -[³H]arginine to l -[³H]citrulline. Incubation with LPS dose-dependently inhibited gap junction permeability to 63.3% at 0.05 µg/ml LPS and no further inhibition was observed on increasing the LPS concentration up to 0.5 µg/ml. LPS-mediated gap junction inhibition was irreversible but was prevented by incubation with the NOS inhibitor NMMA and with the superoxide anion (O₂^??) scavenger superoxide dismutase. Incubation of the cells with both the ^?NO donor S-nitroso-N-acetylpenicillamine and the O₂^??-generating system xanthine/xanthine oxidase inhibited gap junction permeability. These results suggest that the in situ reaction between ^?NO and O₂^??, to form the peroxynitrite anion (ONOO^?), may be responsible for the inhibition of gap junction permeability. Scavenging the ONOO^? derivative hydroxyl radical (^?OH) with either dimethyl sulfoxide or mannitol prevented the LPS-mediated inhibition of gap junction permeability. Finally, exposure of astrocytes to authentic ONOO^? caused a dose-dependent inhibition of gap junction permeability (65.7% of inhibition at 0.5 mM ONOO^?). The pathophysiological relevance of ONOO^?-mediated inhibition of gap junctional communication in astrocytes after NOS induction by LPS is discussed, stressing the possible role played by this mechanism in some neurodegenerative diseases.     

Gap Junction Coupling and Apoptosis in GFSHR-17 Granulosa Cells

Recently, we found that intracellular washout of cGMP induces gap junction uncoupling and proposed a link between gap junction uncoupling and stimulation of apoptotic reactions in GFSHR-17 granulosa cells. In the present report we show that an inhibitor of guanylyl cyclase, ODQ, reduces gap junction coupling and promotes apoptotic reactions such as chromatin condensation and DNA strand breaks. To analyze whether gap junction uncoupling and induction of apoptotic reactions are related, the cells were treated with heptanol and 18β-GA, two known gap junction uncouplers. Gap junction coupling of GFSHR-17 cells could be restored if the incubation time with the gap junction uncouplers was less than 10 min. A prolonged incubation time irreversibly suppressed gap junction coupling and caused chromatin condensation as well as DNA degradation. The promotion of apoptotic reactions by heptanol or 18β-GA was not observed in cells with low gap junction coupling like HeLa cells, indicating that the observed genotoxic reactions are not caused by unspecific effects of gap junction uncouplers. Additionally, it was observed that heptanol or 18β-GA did not induce a sustained rise of [Ca²⁺]_i. The effects of gap junction uncouplers could not be suppressed by the presence of 8-Br-cGMP. It is discussed that irreversible gap junction uncoupling can be mediated by cGMP-dependent as well as cGMP-independent pathways and in turn could lead to stimulation of apoptotic reactions in granulosa cells.     

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size.     

Some Oculodentodigital Dysplasia-Associated Cx43 Mutations Cause Increased Hemichannel Activity in Addition to Deficient Gap Junction Channels

Oculodentodigital dysplasia (ODDD) is a dominantly inherited human disorder associated with different symptoms like craniofacial anomalies, syndactyly and heart dysfunction. ODDD is caused by mutations in the GJA1 gene encoding the gap junction protein connexin43 (Cx43). Here, we have characterized four Cx43 mutations (I31M, G138R, G143S and H194P) after stable expression in HeLa cells. In patients, the I31M and G138R mutations showed all phenotypic characteristics of ODDD, whereas G143S did not result in facial abnormalities and H194P mutated patients exhibited no syndactylies. In transfected HeLa cells, these mutations led to lack of the P2 phosphorylation state of the Cx43 protein, complete inhibition of gap junctional coupling measured by neurobiotin transfer and increased hemichannel activity. In addition, altered trafficking and delayed degradation were found in these mutants by immunofluorescence and pulse-chase analyses. In G138R and G143S mutants, the increased hemichannel activity correlated with an increased half-time of the Cx43 protein. However, the I31M mutated protein showed no extended half-time. Thus, the increased hemichannel activity may be directly caused by an altered conformation of the mutated channel forming protein. We hypothesize that increased hemichannel activity may aggravate the phenotypic abnormalities in ODDD patients who are deficient in Cx43 gap junction channels. Radoslaw Dobrowolski and Annette Sommershof contributed equally to this work.     

Astroglial Gap Junction Communication Is Increased by Treatment with Either Glutamate or High K+ Concentration

Abstract: Astroglia are extensively coupled by gap junctions and form a functional syncytium. Astroglial gap junctions are thought to be involved in the spatial buffering of K⁺ in vivo and in the Ca²⁺ waves seen on glutamate receptor activation. The conductivity of gap junctions is regulated by several second messengers, with up-regulation by cyclic AMP and down-regulation through activation of protein kinase C, decreases in intracellular pH, or increases in the free cytosolic Ca²⁺ concentration. The results presented here indicate that dye coupling of astroglia is significantly up-regulated by membrane depolarization, both by increases in the extracellular K⁺ concentration and directly by ionophores. Furthermore, glutamate, kainate, and quisqualate, which depolarize astroglial cells through activation of ionotropic receptors, also increase dye coupling in astroglia. The effect of kainate and quisqualate was reversed by 6-cyano-7-nitroquinoxaline-2,3-dione, an inhibitor of the ionotropic glutamate receptor. A dose-dependent decrease in dye coupling was seen when the cells were injected with increasing concentrations of Ca²⁺. However, if the cells were simultaneously depolarized, the inhibitory effect of Ca²⁺ on gap junctional conductance was reversed. Significant increases over basal coupling was attained when the cells were injected with Ca²⁺ if they were treated with kainate or K⁺. These data suggest that ligands that depolarize astroglia enhance gap junction communication between astroglia and that this enhancement may be important in maintaining communication between astroglia in the face of elevated Ca²⁺ levels.     

Remodeling of Gap Junctions in Ischemic and Nonischemic Forms of Heart Disease

Electrical activation of the myocardium to produce effective pumping of blood depends on the orderly coordinated spatial and temporal transfer of current from one cell to another via gap junctions. Normal ventricular myocytes are extensively coupled by gap junctions and have the capacity to rapidly increase the amount of connexin within gap junction plaques to meet physiological demands for enhanced cell-cell communication. However, myocytes can also rapidly uncouple in response to injury or disease. In general, both acute and chronic forms of heart disease caused by diverse etiologies are associated with changes in the expression of connexins and remodeling of gap junctions. Such remodeling may have both adaptive and maladaptive consequences and contribute to major clinical processes such as heart failure and sudden cardiac death. Our laboratory has investigated mechanisms regulating cell-cell electrical coupling in the heart under physiological and pathophysiological conditions. This review is focused on selected aspects of this work pertaining to changes in coupling in response to acute and chronic ischemic heart disease and in familial cardiomyopathies caused by mutations in genes encoding desmosomal proteins.     

Unusual Slow Gating of Gap Junction Channels in Oocytes Expressing Connexin32 or Its COOH-Terminus Truncated Mutant

Gap junction channels are gated by a chemical gate and two transjunctional voltage (V _j)-sensitive gates: fast and slow. Slow V _j gate and chemical gate are believed to be the same. The slow gate closes at the negative side of V _j and is mostly inactive without uncouplers or connexin (Cx) mutations. In contrast, our present data indicate otherwise. Oocytes expressing Cx32 were subjected to series of −100 mV V _j pulses (12-s duration, 30-s intervals). Both peak (PK) and steady-state (SS) junctional conductances (G _j), measured at each pulse, decreased exponentially by 50−60% (tau = ∼1.2 min). G _jPK dropped more dramatically, such that G _jSS/G _jPK increased from 0.4 to 0.6, indicating a drop in V _j sensitivity. Less striking effects were obtained with –60 mV pulses. During recovery, G _j, measured by applying 20 mV pulses (2-s duration, 30-s intervals), slowly returned to initial values (tau = ∼7 min). With reversal of V _j polarity, G _jPK briefly increased and G _jSS/G _jPK decreased, suggesting that V _j-dependent hemichannel reopening is faster than hemichannel closing. Similar yet more dramatic results were obtained with COOH-terminus truncated Cx32 (Cx32-D225), a mutant believed to lack fast V _j gating. The data indicate that the slow gate of Cx32 is active in the absence of uncouplers or mutations and displays unusual V _j behavior. Based on previous evidence for direct calmodulin (CaM) involvement in chemical/slow gating, this may also be CaM-mediated.     

Quantitative Determination of Gap Junction Intercellular Communication by Scrape Loading and Image Analysis

Gap junction intercellular communication (GJIC) consists of intercellular exchange of low molecular weight molecules. Chemically induced alterations of this communication have been suggested to result in abnormal cell growth and tumour promotion. Several in vitro assays have been developed to determine the effect of chemicals on gap junction communication in cultured cells. The scrape loading dye transfer technique is based on studying the transfer of the fluorescent dye Lucifer Yellow in cells where the dye is loaded through a cut in the cell monolayer. This technique is rapid and relatively uncomplicated, but has only been used to qualitatively demonstrate communication, due to lack of an appropriate method for quantification of the dye spreading. We show here that analysis of digital fluorescence images of cells scrape loaded with Lucifer Yellow can be used for quantitative determination of GJIC. We have analysed the images both by means of distance of diffusion of the dye in the cell monolayer, as well as by area of dye-coupled cells. The results are consistent with that obtained using microinjection of Lucifer Yellow and the method offers a simple way for quantitative determination of GJIC.     

Identification and Functional Expression of HCx31.9, a Novel Gap Junction Gene

By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.     

Loss of Gap Junction Plaques and Inhibition of Intercellular Communication in Ilimaquinone-treated BICR-M1Rk and NRK Cells

To examine the mechanism(s) and pathways of gap junction formation and removal a novel and reversible inhibitor of protein secretion, ilimaquinone (IQ), was employed. IQ has been reported to cause the vesiculation of Golgi membranes, block protein transport at the cis-Golgi and depolymerize cytoplasmic microtubules. Connexin43 (Cx43) immunolabeling and dye microinjection experiments revealed that gap junction plaques were lost and intercellular communication was inhibited following IQ treatment for 1 hr in BICR-M1R_k rat mammary tumor cells and for 2 hr in normal rat kidney (NRK) cells. Gap junction plaques and intercellular communication recovered within 2 hr when IQ was removed. IQ, however, did not affect the distribution of zonula occludens-1, a protein associated with tight junctions. Western blot analysis revealed that the IQ-induced loss of gap junction plaques was accompanied by a limited reduction in the highly phosphorylated form of Cx43, previously shown to be correlated with gap junction plaques. The presence of IQ inhibited the formation of new gap junction plaques in BICR-M1R_k cells under conditions where preexisting gap junctions were downregulated by brefeldin A treatment. Treatment of BICR-M1R_k and NRK cells with other microtubule depolymerization agents did not inhibit plaque formation or promote rapid gap junction removal. These findings suggest that IQ disrupts intercellular communication by inhibiting the events that are involved in plaque formation and/or retention at the cell surface independent of its effects on microtubules. Our results also suggest that additional factors other than phosphorylation are necessary for Cx43 assembly into gap junction plaques. Received: 16 January 1996/Revised: 20 September 1996     

A History of Gap Junction Structure: Hexagonal Arrays to Atomic Resolution

Abstract

Gap junctions are specialized membrane structures that provide an intercellular pathway for the propagation and/or amplification of signaling cascades responsible for impulse propagation, cell growth, and development. Prior to the identification of the proteins that comprise gap junctions, elucidation of channel structure began with initial observations of a hexagonal nexus connecting apposed cellular membranes. Concomitant with technological advancements spanning over 50 years, atomic resolution structures are now available detailing channel architecture and the cytoplasmic domains that have helped to define mechanisms governing the regulation of gap junctions. Highlighted in this review are the seminal structural studies that have led to our current understanding of gap junction biology.     

Conductive and Kinetic Properties of Connexin45 Hemichannels Expressed in Transfected HeLa Cells

Human HeLa cells transfected with mouse connexin Cx45 were used to examine the conductive and kinetic properties of Cx45 hemichannels. The experiments were carried out on single cells using a voltage-clamp method. Lowering the [Ca²⁺]_o revealed an extra current. Its sensitivity to extracellular Ca²⁺ and gap junction channel blockers (18α-glycyrrhetinic acid, palmitoleic acid, heptanol), and its absence in non-transfected HeLa cells suggested that it is carried by Cx45 hemichannels. The conductive and kinetic properties of this current, I _hc, were determined adopting a biphasic pulse protocol. I _hc activated at positive V _m and deactivated partially at negative V _m. The analysis of the instantaneous I _hc yielded a linear function g _hc,inst = f(V _m) with a hint of a negative slope (g _hc,inst: instantaneous conductance). The analysis of the steady-state I _hc revealed a sigmoidal function g _hc,ss = f(V _m) best described with the Boltzmann equation: V _m,0 = −1.08 mV, g _hc,min = 0.08 (g _hc,ss: steady-state conductance; V _{m, 0}:V _m at which g _hc,ss is half-maximally activated; g _hc,min: minimal conductance; major charge carriers: K⁺ and Cl⁻). The g _hc was minimal at negative V _m and maximal at positive V _m. This suggests that Cx45 connexons integrated in gap junction channels are gating with negative voltage. I _hc deactivated exponentially with time, giving rise to single time constants, τ_d. The function τ_d = f(V _m) was exponential and increased with positive V _m (τ_d = 7.6 s at V _m = 0 mV). The activation of I _hc followed the sum of two exponentials giving rise to the time constants, τ_a1 and τ_a2. The function τ_a1 = f(V _m) and τ_a2 = f(V _m) were bell-shaped and yielded a maximum of ≅ 0.6 s at V _m ≅ −20 mV and ≅ 4.9 s at V _m ≅ 15 mV, respectively. Neither τ_a1 = f(V _m) nor τ_a2 = f(V _m) coincided with τ_d = f(V _m). These findings conflict with the notion that activation and deactivation follow a simple reversible reaction scheme governed by first-order voltage-dependent processes.     

Gap junctions suggest epithelial conduction within the comb plates of the ctenophore Pleurobrachia bachei

Intercellular gap junctions occur between the ciliated cells that make up the comb plates of the ctenophore Pleurobrachia. Similar junctions are found within the ciliated grooves which run from the apical organ to the first plate of each comb row, as well as throughout the endoderm of the meridional canals. Gap junctions were not found in the ectodermal tissue between the comb rows. The distribution of junctions suggests that excitation conduction within the ciliated grooves, comb plates and meridional canal endoderm may be epithelial.     

Gap junctions in the cardiac muscle cells of the lamprey

Summary Electron microscopy of both thin sections and freeze-fracture replicas has demonstrated the occurrence of gap junctions (nexuses) in the cardiac muscle cells of the lamprey. These gap junctions are identical in basic structure with those found in the mammalian heart. However, they are much smaller (less than 0.5 in diameter), and more irregularly distributed than the typical gap junction in the mammalian heart. These small gap junctions seem to provide a structural basis for the electrical coupling between cardiac muscle cells in the lower vertebrates.In addition, the well developed sarcoplasmic reticulum and subsurface cisternae, which contain an electron dense spheroidal cast, are frequently observed in the cardiac muscles of the lamprey.This work is supported by a research grant from the Ministry of Education, Japan