间隙连接蛋白43突变与皮肤疾病相关性研究

间隙连接蛋白(Cx)在保证细胞间物质信息传递及维持皮肤屏障稳态方面发挥着重要作用,其基因突变,甚至表达水平异常均会引起多种疾病,严重影响患者生活质量。在编码人类Cx家族的21个基因中,与Cx基因突变相关的临床伴随疾病至少有14种。其中,Cx43分布最广,不仅在大多数器官组织中均有报道,也是伤口愈合、皮肤角质化,以及皮肤肿瘤发展等重要生理病理过程中的关键调控节点。本文总结了近年来Cx43基因(GJA1)在皮肤屏障中的作用、GJA1基因突变相关皮肤疾病及其潜在致病机制这几个快速发展领域中的研究成果,以期为Cx43临床伴发疾病防治及相关研究提供参考。     

Differential connexin expression,gap junction intercellular coupling,and hemichannel formation in NT2/D1 human neural progenitors and terminally differentiated hNT neurons

Connexin-mediated gap junctions and open hemichannels in nonjunctional membranes represent two biologically relevant mechanisms by which neural progenitors can coordinate their response to changes in the extracellular environment. NT2/D1 cells are a teratocarcinoma progenitor line that can be induced to differentiate terminally into functional hNT neurons and NT-G nonneuronal cells. Clinical transplants of hNT neurons and experimental grafts of NT2/D1 progenitors or hNT neurons have been used in cell-replacement therapy in vivo. Previous studies have shown that NT2/D1 cells express connexin 43 (Cx43) and that NT2/D1 progenitors are capable of dye transfer. To determine whether NT2/D1 progenitors and differentiated hNT cultures express other connexins, Cx26, Cx30, Cx32, Cx36, Cx37, Cx43, and Cx46.6 mRNA and protein were analyzed. NT2/D1 progenitors express Cx30, Cx36, Cx37, and Cx43. hNT/NT-G cultures express Cx36, Cx37, and de novo Cx46.6. Cx26 and Cx32 were not expressed in NT2/D1 or hNT/NT-G cells. NT2/D1 progenitors formed functional gap junctions as assessed by dye coupling as well as open hemichannels in nonjunctional membranes as assessed by dye-uptake studies. Dye coupling was inhibited by the gap junction blocker 18alpha-glycyrrhetinic acid. Hemichannel activity was inhibited by the dual-specificity chloride channel/connexin hemichannel inhibitor flufenamic acid but not by the chloride channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Both dye coupling and dye uptake were substantially reduced following differentiation of NT2/D1 progenitors. We conclude that the pattern of connexin expression in NT2/D1 cells changes over the course of differentiation corresponding with a reduction in biochemical coupling and hemichannel activity in differentiated cells.     

Prenatal exposure to inflammatory conditions increases Cx43 and Panx1 unopposed channel opening and activation of astrocytes in the offspring effect on neuronal survival

Several epidemiological studies indicate that children born from mothers exposed to infections during gestation, have an increased risk to develop neurological disorders, including schizophrenia, autism and cerebral palsy. Given that it is unknown if astrocytes and their crosstalk with neurons participate in the above mentioned brain pathologies, the aim of this work was to address if astroglial paracrine signaling mediated by Cx43 and Panx1 unopposed channels could be affected in the offspring of LPS‐exposed dams during pregnancy. Ethidium uptake experiments showed that prenatal LPS‐exposure increases the activity of astroglial Cx43 and Panx1 unopposed channels in the offspring. Induction of unopposed channel opening by prenatal LPS exposure depended on intracellular Ca²⁺ levels, cytokine production and activation of p38 MAP kinase/iNOS pathway. Biochemical assays and Fura‐2AM/DAF‐FM time‐lapse fluorescence images revealed that astrocytes from the offspring of LPS‐exposed dams displayed increased spontaneous Ca²⁺ dynamics and NO production, whereas iNOS levels and release of IL‐1β/TNF‐α were also increased. Interestingly, we found that prenatal LPS exposure enhanced the release of ATP through astroglial Cx43 and Panx1 unopposed channels in the offspring, resulting in an increased neuronal death mediated by the activation of neuronal P2X₇ receptors and Panx1 channels. Altogether, this evidence suggests that astroglial Cx43 and Panx1 unopposed channel opening induced by prenatal LPS exposure depended on the inflammatory activation profile and the activation pattern of astrocytes. The understanding of the mechanism underlying astrocyte‐neuron crosstalk could contribute to the development of new strategies to ameliorate the brain abnormalities induced in the offspring by prenatal inflammation. GLIA 2015;63:2058–2072     

次声刺激诱导原代培养大鼠星形胶质细胞释放谷氨酸的研究

摘要 目的：观察16Hz, 130dB次声刺激对原代培养的大鼠星形胶质细胞谷氨酸释放的影响,并探讨其释放机制。 方法：原代培养大鼠海马区星形胶质细胞,将其分为次声刺激组（IE group,n=6）,Gap26+次声刺激组（Gap26+IE group）,对照组（Ctrl group,n=6）。次声刺激组细胞暴露于次声舱中,分别给予15min, 30min, 60min, 90min, 120min和240min的次声刺激;对于Gap26+IE组,则在次声刺激前,选用Cx43半通道阻断剂Gap26预处理星形胶质细胞;对照组也置于次声舱,但不给予次声刺激。次声刺激频率为16Hz,强度为130dB。为了排除Gap26对谷氨酸释放的影响,还选用乱序Gap26肽段(Scrb Gap26)来预处理星形胶质细胞（Scrb Gap26+IE group,n=6）。采用免疫荧光染色测定细胞Cx43的表达情况,采用高效液相色谱（HPLC）来测定细胞外液谷氨酸浓度。 结果：次声刺激后,IE组细胞外液谷氨酸的浓度显著升高,并在刺激90min后,细胞外液谷氨酸浓度达峰值,为（4.6±0.3）nmol/ml,显著高于对照组（2.3±0.2）nmol/ml（P<0.05）,这说明次声刺激可以显著增高星形胶质细胞外液的谷氨酸含量。此外,次声刺激后,IE组细胞Cx43的表达也显著升高,刺激60mins时,Cx43平均荧光强度达到峰值,为（198±33）(AUC),显著高于对照组（P<0.05）。而对于Gap26+IE组,次声刺激后细胞外液谷氨酸浓度的升高受到抑制,90min后,细胞外液谷氨酸浓度为（3.58±0.17）nmol/ml,显著低于次声刺激组（P<0.05）。 结论：次声刺激可以诱导星形胶质细胞释放谷氨酸;Cx43半通道阻断剂Gap26抑制了谷氨酸释放,次声刺激诱导的谷氨酸释放可能是通过Cx43半通道来完成的。     

Biological responses of osteocytic connexin 43 hemichannels to simulated microgravity

Connexin 43 (Cx43) hemichannels and gap junctions in osteocytes are responsive to mechanical loading, which is important for bone formation and remodeling. However, the mechanism of these Cx43‐forming channels in the process of mechanical unloading is still not very clear. In this study, unloading caused by weightlessness was simulated by using a random position machine (RPM). Osteocytic MLO‐Y4 cells were subjected to 2 h of RPM treatment, and levels of Cx43 mRNA and total and cell surface expressed protein were determined by quantitative real‐time PCR, western blotting, and biotinylation analysis. Although mRNA was elevated by RPM, total protein level of Cx43 was not altered; however, surface biotinylated Cx43 was significantly reduced. Interestingly, RPM promoted the retention of Cx43 in the Golgi apparatus detected by co‐immunofluorescence with antibodies against Cx43 and 58 K Golgi marker protein. Dye uptake assay showed that hemichannels were induced open after RPM for 2 h. Consistently, prostaglandin E₂ release was increased and this increase was completely attenuated with the treatment of a Cx43 hemichannel blocking antibody. Together, this study demonstrates increased activity of Cx43 hemichannels to RPM, and active Cx43 hemichannels with prostaglandin E₂ release are likely to module biological function under simulated weightless conditions. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1195–1202, 2017.

     

Differential susceptibility of Cx26 mutations associated with epidermal dysplasias to peptidoglycan derived from Staphylococcus aureus and Staphylococcus epidermidis

Mutations in Connexin26 (Cx26) give rise to a spectrum of dominantly inherited hyperproliferating skin disorders, the severest being keratitis–ichthyosis–deafness (KID) syndrome, an inflammatory skin disorder, with patients prone to opportunistic infections. We compared the effects of peptidoglycan (PGN) extracted from the skin commensal Staphylococcus epidermidis and the opportunistic pathogen Staphylococcus aureus on interleukin‐6 and connexin expression in HaCaT cells (a keratinocyte cell line) and connexin channel activity in HaCaT and HeLa (connexin deficient) cells transfected to express KID and non‐KID Cx26 mutations. In both cell types, PGN from S. aureus induced hemichannel activity in cells expressing KID mutants as monitored by ATP release assays following 15‐min challenge, while that from S. epidermidis evoked a response in HeLa cells. In KID mutant expressing cells, ATP release was significantly higher than in cells transfected with wild‐type Cx26. No ATP release was observed in non‐KID mutant transfected cells or in the presence of carbenoxolone, a connexin channel blocker. PGN isolated from S. aureus but not S. epidermidis induced interleukin‐6 and Cx26 expression in HaCaT cells following 6‐h challenge. Challenge by PGN from S. aureus evoked a greater interleukin‐6 response in cells expressing KID mutants than in cells expressing wtCx26 or non‐KID mutants. This response returned to basal levels if acute KID hemichannel signalling was blocked prior to PGN challenge. Thus, KID mutants form channels that can be triggered by the pro‐inflammatory mediator PGN from opportunistic pathogens but not skin commensals, providing further insight into the genotype–phenotype relationship of Cx26 disorders.     

Retinal horizontal cell-specific promoter activity and protein expression of zebrafish connexin 52.6 and connexin 55.5

Connexins in retinal horizontal cells (HC) function in the processing of visual information. For example, gap junction-forming connexins may contribute to the spatial integration of visual stimuli. Additionally, connexin hemichannels have been hypothesized to participate in the feedback pathway from HCs to cones. To verify the identities of the zebrafish HC connexins, we performed promoter expression and immunohistochemical studies of connexin 52.6 (Cx52.6) and Cx55.5. Zebrafish embryos were microinjected with Cx52.6 or Cx55.5 promoter sequences and a green fluorescent protein reporter construct. Light and electron microscopic (EM) analysis showed green fluorescent protein expression exclusively in retinal HCs. Immunohistochemistry confirmed that HCs express Cx52.6 and Cx55.5 proteins. Light microscopy revealed Cx52.6 and Cx55.5 in the retinal inner nuclear and outer plexiform layers. Double labeling for Cx55.5 or Cx52.6 and cell-specific markers (tyrosine hydroxylase, protein kinase C-alpha, or GluR2) demonstrated that these connexins do not localize to interplexiform or ON bipolar cells, but most likely are present in HCs. Preembedding immuno-EM confirmed the HC-specific expression of Cx52.6 and Cx55.5 and illustrated the presence of these two connexins in gap junctions between HCs. The EM data also revealed robust labeling for Cx55.5 in hemichannels on HC dendrites in photoreceptor synaptic terminals. Voltage-clamp experiments in cultured cells demonstrated that Cx55.5-containing hemichannels can open at physiological membrane potentials. These results offer the first in vivo demonstration of the HC-specific activities of the Cx52.6 and Cx55.5 promoters. Furthermore, these data provide the first proof at the protein level for retinal HC-specific connexins in the zebrafish.     

Localization of the pannexin1 protein at postsynaptic sites in the cerebral cortex and hippocampus

Pannexins (Panx) constitute a new family of gap junction type proteins. Functional expression in paired Xenopus oocytes indicated that pannexins are capable of forming communicating junctions but also proved to be active in forming of unopposed hemichannels. In the vertebrate brain pannexins have been found in neurons. However, the subcellular cerebral localization of pannexin proteins which could gain first clues on their putative function is essentially unknown. Here we demonstrate by light and electron microscopical immunohistochemistry that Panx1 reveals postsynaptic localization in rodent hippocampal and cortical principal neurons accumulating at postsynaptic densities. The postsynaptic localization was corroborated by co-localization of Panx1 with postsynaptic density protein 95 (PSD-95), a prominent postsynaptic scaffolding protein, in hippocampal neurons expressing tagged versions of these proteins. The asymmetric synaptic distribution of Panx1 suggests that it may function in neurons as non-junctional channels (pannexons) at postsynaptic sites and comprises a novel component of the postsynaptic protein complex.     

Targeting connexin hemichannels to control the inflammasome: the correlation between connexin43 and NLRP3 expression in chronic eye disease

ABSTRACT

Introduction: Chronic inflammatory diseases, including retinal diseases that are a major cause of vision loss, are associated with activation of the nucleotide-binding domain and leucine-rich repeat containing (NLR) protein-3 (NLRP3) inflammasome pathway. In chronic disease, the inflammasome becomes self-perpetuating, indicating a common pathway in such diseases irrespective of underlying etiology, and implying a shared solution is feasible. Connexin43 hemichannels correlate directly with NLRP3 inflammasome complex assembly (shown here in models of retinal disease). Connexin43 hemichannel-mediated ATP release is proposed to be the principal activator signal for inflammasome complex assembly in primary signal-sensitized cells. Connexin hemichannel block on its own is sufficient to inhibit the inflammasome pathway.

Areas covered: We introduce chronic retinal disease, discuss available preclinical models and examine findings from these models regarding the targeting of connexin43 hemichannels and its effects on the inflammasome.

Expert opinion: In over 25 animal disease models, connexin hemichannel regulation has shown therapeutic benefit, and one oral connexin hemichannel blocker, tonabersat (Xiflam), is Phase II ready with safety evidence in over 1000 patients. Regulating the connexin hemichannel provides a means to move quickly into clinical trials designed to ameliorate the progression of devastating chronic diseases of the eye, but also elsewhere in the body.