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.     

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.     

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.     

Gap junctional regulation of signal transduction in bone cells

The role of gap junctions, particularly that of connexin43 (Cx43), has become an area of increasing interest in bone physiology. An abundance of studies have shown that Cx43 influences the function of osteoblasts and osteocytes, which ultimately impacts bone mass acquisition and skeletal homeostasis. However, the molecular details underlying how Cx43 regulates bone are only coming into focus and have proven to be more complex than originally thought. In this review, we focus on the diverse molecular mechanisms by which Cx43 gap junctions and hemichannels regulate cell signaling pathways, gene expression, mechanotransduction and cell survival in bone cells. This review will highlight key signaling factors that have been identified as downstream effectors of Cx43 and the impact of these pathways on distinct osteoblast and osteocyte functions.     

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。     

Roles of Gap Junctions and Connexins in Non-Neoplastic Pathological Processes in which Cell Proliferation Is Involved

Cell proliferation is an important process for reproduction, growth and renewal of living cells and occurs in several situations during life. Cell proliferation is present in all the steps of carcinogenesis, initiation, promotion and progression. Gap junctions are the only specialization of cell membranes that allows communication between adjacent cells. They are known to contribute to tissue homeostasis and are composed of transmembrane proteins called “connexins.” These junctions are also known to be involved in cell proliferation control. The roles of gap junctions and connexins in cell proliferation are complex and still under investigation. Since pioneer studies by Loewenstein, it is known that neoplastic cells lack communicating junctions. They do not communicate with their neighbors or with non-neoplastic cells from the surrounding area. There are many studies and review articles dedicated to neoplastic tissues. The aim of this review is to present evidence on the roles of gap junctions and connexins in non-neoplastic processes in which cell proliferation is involved.     

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.     

Gap junction regulation by calmodulin

Intracellular Ca²⁺ activated calmodulin (CaM) inhibits gap junction channels in the low nanomolar to high micromolar range of [Ca²⁺]_i. This regulation plays an essential role in numerous cellular processes that include hearing, lens transparency, and synchronized contractions of the heart. Previous studies have indicated that gap junction mediated cell-to-cell communication was inhibited by CaM antagonists. More recent evidence indicates a direct role of CaM in regulating several members of the connexin family. Since the intracellular loop and carboxyl termini of connexins are largely “invisible” in electron microscopy and X-ray crystallographic structures due to disorder in these domains, peptide models encompassing the putative CaM binding sites of several intracellular domains of connexins have been used to identify the Ca²⁺-dependent CaM binding sites of these proteins. This approach has been used to determine the CaM binding affinities of peptides derived from a number of different connexin-subfamilies.     

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.     

Gap Junctions and Cochlear Homeostasis

Gap junctions play a critical role in hearing and mutations in connexin genes cause a high incidence of human deafness. Pathogenesis mainly occurs in the cochlea, where gap junctions form extensive networks between non-sensory cells that can be divided into two independent gap junction systems, the epithelial cell gap junction system and the connective tissue cell gap junction system. At least four different connexins have been reported to be present in the mammalian inner ear, and gap junctions are thought to provide a route for recycling potassium ions that pass through the sensory cells during the mechanosensory transduction process back to the endolymph. Here we review the cochlear gap junction networks and their hypothesized role in potassium ion recycling mechanism, pharmacological and physiological gating of cochlear connexins, animal models harboring connexin mutations and functional studies of mutant channels that cause human deafness. These studies elucidate gap junction functions in the cochlea and also provide insight for understanding the pathogenesis of this common hereditary deafness induced by connexin mutations. H.-B. Zhao, T. Kikuchi, A. Ngezahayo, T. W. White contributed equally to this article     

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     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

外胚层细胞诱导后的间隙连接

用电镜观察了经受诱导作用之后胚胎细胞的冷冻蚀刻复型膜。和未经诱导作用的对照组比较,早期和中期神经胚的神经上皮细胞以及经过豚鼠骨髓粗提液(BME)——一种有效的异源中胚层诱导物——处理过的早期原肠胚外胚层,它们的间隙连接都处于活跃的动态状态。用图像分析仪测得的间隙连接连接子的排列密度,指出经受过诱导作用的三组分别和未经受诱导作用的对照组比较,计算求出P值,神经上皮两组和对照组的差别为非常显著,BME处理过的细胞和对照组的差别为显著。结合对照组与诱导后胚胎细胞间隙连接连接子的变化讨论了它们在信息传递上可能起的作用。     

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.     

Gap junctional communication in tissue inflammation and repair

Local injury induces a complex orchestrated response to stimulate healing of injured tissues, cellular regeneration and phagocytosis. Practically, inflammation is defined as a defense process whereby fluid and white blood cells accumulate at a site of injury. The balance of cytokines, chemokines, and growth factors is likely to play a key role in regulating important cell functions such as migration, proliferation, and matrix synthesis during the process of inflammation. Hence, the initiation, maintenance, and resolution of innate responses depend upon cellular communication. A process similar to tissue repair and subsequent scarring is found in a variety of fibrotic diseases. This may occur in a single organ such as liver, kidneys, pancreas, lung, skin, and heart, but fibrosis may also have a more generalized distribution such as in atherosclerosis. The purpose of this review is to summarize recent advances on the contribution of gap junction-mediated intercellular communication in the modulation of the inflammatory response and tissue repair.     

Changes of Gap and Tight Junctions during Differentiation of Human Nasal Epithelial Cells Using Primary Human Nasal Epithelial Cells and Primary Human Nasal Fibroblast Cells in a Noncontact Coculture System

The epithelium of upper respiratory tissues such as nasal mucosa forms a continuous barrier to a wide variety of exogenous antigens. The epithelial barrier function is regulated in large part by the intercellular junctions, referred to as gap and tight junctions. However, changes of gap and tight junctions during differentiation of human nasal epithelial (HNE) cells are still unclear. In the present study, to investigate changes of gap and tight junctions during differentiation of HNE cells in vitro, we used primary human HNE cells cocultured with primary human nasal fibroblast (HNF) cells in a noncontact system. In HNE cells cocultured with HNF cells for 2 weeks, numerous elongated cilia-like structures were observed compared to those without HNF cells. In the coculture, downregulation of Cx26 and upregulation of Cx30.3 and Cx31 were observed together with extensive gap junctional intercellular communication. Furthermore, expression of the tight junction proteins claudin-1, claudin-4, occludin and ZO-2 was increased. These results suggest that switching in expression of connexins and induction of tight junction proteins may be closely associated with differentiation of HNE cells in vitro and that differentiation of HNE cells requires unknown soluble factors secreted from HNF cells.