Connexin Research Articles

Effect of platelet-derived growth factor-BB on gap junction and connexin43 in rat penile corpus cavernosum smooth muscle cells.

We explored whether platelet-derived growth factor (PDGF)-BB regulates corpus cavernosum smooth muscle cell gap junctions and can ameliorate erectile dysfunction and how it modulates connexin43 (CX43) after bilateral cavernous neurectomy. Primary cultured rat corpus cavernosum smooth muscle cells were treated with PDGF-BB with or without a PDGFR inhibitor, Akt siRNA or the depletion or promotion of β-catenin. PDGF-BB improved CCSMCs gap junction coupling and increased CX43 and PDGFRβ expression; inhibition of PDGFR activity down-regulated CX43 and decreased Akt and nuclear β-catenin. Knockdown or promotion of β-catenin down-regulated and up-regulated CX43 expression respectively. Moreover, β-catenin activation induced CX43 nuclear accumulation, which impeded CX43 down-regulation induced by PDGFR inhibition, suggesting that CX43 expression is positively correlated with nuclear β-catenin expression. Furthermore, CX43 promoter luciferase and chromatin immunoprecipitation assays indicated that β-catenin regulates CX43 transcription by directly interacting with its promoter. Male rats underwent bilateral cavernous neurectomy. After 12weeks, they were injected with PDGF-BB, CX43 and PDGFRβ expression was significantly lower than in the control group, which was reversed by PDGF-BB injection. These results suggested that PDGF-BB contributed to the improvement of gap junction intracellular communication among corpus cavernosum smooth muscle cells, increased CX43 through PDGFRβ/Akt/nuclear β-catenin signalling, and ameliorated cavernous nerve injury-induced erectile dysfunction.

Enhanced Function of Induced Pluripotent Stem Cell-Derived Endothelial Cells Through ESM1 Signaling.

The mortality rate for (cardio)‐vascular disease is one of the highest in the world, so a healthy functional endothelium is of outmost importance against vascular disease. In this study, human induced pluripotent stem (iPS) cells were reprogrammed from 1 ml blood of healthy donors and subsequently differentiated into endothelial cells (iPS‐ECs) with typical EC characteristics. This research combined iPS cell technologies and next‐generation sequencing to acquire an insight into the transcriptional regulation of iPS‐ECs. We identified endothelial cell‐specific molecule 1 (ESM1) as one of the highest expressed genes during EC differentiation, playing a key role in EC enrichment and function by regulating connexin 40 (CX40) and eNOS. Importantly, ESM1 enhanced the iPS‐ECs potential to improve angiogenesis and neovascularisation in in vivo models of angiogenesis and hind limb ischemia. These findings demonstrated for the first time that enriched functional ECs are derived through cell reprogramming and ESM1 signaling, opening the horizon for drug screening and cell‐based therapies for vascular diseases. Therefore, this study showcases a new approach for enriching and enhancing the function of induced pluripotent stem (iPS) cell‐derived ECs from a very small amount of blood through ESM1 signaling, which greatly enhances their functionality and increases their therapeutic potential. Stem Cells 2019;37:226–239

In�vitro study of the effects of reprogramming neonatal rat fibroblasts transfected with TBX18 on spontaneous beating in neonatal rat cardiomyocytes

Intracardiac injection of the growth-promoting transcription factor of the sinoatrial node T-box18 (TBX18) has been shown to reprogram cardiomyocytes into induced sinoatrial nodes that produce high-frequency and neuroregulated ectopic beats. Fibroblasts, the most important non-cardiomyocyte cell type in the heart, can affect the electrophysiological properties of cardiomyocytes by electrically coupling with them. The aim of the present study was to explore the reprogramming of cardiac fibroblasts (CFs) transfected with TBX18 in vitro and observe its effect on the pacing frequency of neonatal rat ventricular cardiomyocytes (NRVMs) in a co-culture system. CFs transfected with TBX18 could be transformed into cardiac myofibroblasts that expressed high levels of hyperpolarization-activated cyclic nucleotide-gated cation channel 4 protein and low levels of connexin 43 (COX43) and COX45 protein. In addition, TBX18-CFs could increase the beating rates of NRVMs and TBX18-NRVMs in a co-culture system. The results of the present study indicated that the TBX18 gene could induce CFs to undergo a transformation that promotes an increase of the beating rates of NRVMs and TBX18-NRVMs.

Connexin 43-Mediated Mitochondrial Transfer of iPSC-MSCs Alleviates Asthma Inflammation.

SummaryWe previously identified an immunomodulatory role of human induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (MSCs) in asthmatic inflammation. Mitochondrial transfer from bone marrow MSCs to epithelial cells can result in the attenuation of acute lung injury in mice. However, the effects of mitochondrial transfer from iPSC-MSCs to epithelial cells in asthma and the mechanisms underlying these effects are unclear. We found that iPSC-MSC transplantation significantly reduced T helper 2 cytokines, attenuated the mitochondrial dysfunction of epithelial cells, and alleviated asthma inflammation in mice. Tunneling nanotubes (TNTs) were formed between iPSC-MSCs and epithelial cells, and mitochondrial transfer from iPSC-MSCs to epithelial cells via TNTs was observed both in vitro and in mice. Overexpression or silencing of connexin 43 (CX43) in iPSC-MSCs demonstrated that CX43 plays a critical role in the regulation of TNT formation by mediating mitochondrial transfer between iPSC-MSCs and epithelial cells. This study provides a therapeutic strategy for targeting asthma inflammation.

Implant abutment microgrooves affect soft tissue cells response via connexin 43 pathway

Background The down-regulation of Connexin 43 (CX43) hemichannels (HCs) function in soft tissue cells promotes the up-regulation of numerous pro-healing genes, as the down-regulation of pro-fibrotic ones, leading to its identification as a potential target for the modulation of wound healing. On the other hand, titanium is commonly used as a transmucosal material, both for dental implants and abutments, and its surface micro-topography is known to affect cell responses. Aim/Hypothesis To investigate whether threaded titanium surfaces with different groove characteristics modulate CX43 expression and its organization in HCs rather than in gap junction (GJs) in an immortalized mouse myoblast cell line. In addition, to verify if the growth pattern and arrangement of cells on the grooves. Material and Methods Murine myoblasts C2C12 were seeded on discs with grooved titanium surfaces with different patterns- UTM (Ultrathin Threaded Microsurface) and XA microgrooved surface. A machined surface was used as control (MA). Titanium surface specimens were kindly provided by Sweden & Martina (Padova, Italy). Cells were cultured up to 7 days and daily monitored for viability through chemiluminescence (cell proliferation). Gene expression analysis for CX43, alkaline phosphatase (ALP) and collagen 1 (COL1a) was performed+ after 3 days of culture total RNA was purified, retro-transcribed to cDNA and amplified through quantitative RT-PCR. To localize the expression of CX43 and thus to identify its organization as HCs or as GJs, cells were observed under fluorescence after actin, CX43 and nuclei labeling. Differences among the groups were evaluated through Two-Way ANOVA statistical test. Results Very weak signal for CX43 was observed on UTM surface (mRNA 6.12-fold less than XA and 2.58-fold less than MA). CX43 expression was extremely up-regulated on XA surface after 3 days, when compared to UTM and MA surface (P < 0.0001). Conversely, cell proliferation was significantly reduced (P < 0.0001). On the XA surface, CX43 expression was labelled in the peri-nuclear area, indicating its organization in HCs. MA showed a slightly enhanced level of CX43 (2.37-fold less than XA) and, where cells were confluent, CX43 signal was marked around cell nuclei (HCs) and at the edges of cell profile, indicating GJs with other cells. Noteworthy, the disposition of cells on XA and UTM surfaces followed the grid of the grooved surface topography, with cells laying on the grid hollows and elongated to colonize the surface. Furthermore, gene expression analysis revealed that MA control surface induced a commitment of C2C12 towards an osteoblastic phenotype, showing greater levels of ALP and COL1a. Conclusions and Clinical Implications The microgrooved titanium surfaces showed a modulatory role on myoblasts, promoting an evident cell arrangement along the surface grooves on UTM and XA surfaces. This may lead in vivo to the deposition of an oriented extracellular matrix. Additionally, UTM surface caused CX43 down-regulation, leading to the hypothesis of a more favorable in vivo soft tissues wound healing.

Fast Generation of Functional Subtype Astrocytes from Human Pluripotent Stem Cells.

SummaryDifferentiation of astrocytes from human pluripotent stem cells (hPSCs) is a tedious and variable process. This hampers the study of hPSC-generated astrocytes in disease processes and drug development. By using CRISPR/Cas9-mediated inducible expression of NFIA or NFIA plus SOX9 in hPSCs, we developed a method to efficiently generate astrocytes in 4–7 weeks. The astrocytic identity of the induced cells was verified by their characteristic molecular and functional properties as well as after transplantation. Furthermore, we developed a strategy to generate region-specific astrocyte subtypes by combining differentiation of regional progenitors and transgenic induction of astrocytes. This simple and efficient method offers a new opportunity to study the fundamental biology of human astrocytes and their roles in disease processes.

A Cell Junctional Protein Network Associated with Connexin-26.

GJB2 mutations are the leading cause of non-syndromic inherited hearing loss. GJB2 encodes connexin-26 (CX26), which is a connexin (CX) family protein expressed in cochlea, skin, liver, and brain, displaying short cytoplasmic N-termini and C-termini. We searched for CX26 C-terminus binding partners by affinity capture and identified 12 unique proteins associated with cell junctions or cytoskeleton (CGN, DAAM1, FLNB, GAPDH, HOMER2, MAP7, MAPRE2 (EB2), JUP, PTK2B, RAI14, TJP1, and VCL) by using mass spectrometry. We show that, similar to other CX family members, CX26 co-fractionates with TJP1, VCL, and EB2 (EB1 paralogue) as well as the membrane-associated protein ASS1. The adaptor protein CGN (cingulin) co-immuno-precipitates with CX26, ASS1, and TJP1. In addition, CGN co-immunoprecipitation with CX30, CX31, and CX43 indicates that CX association is independent on the CX C-terminus length or sequence. CX26, CGN, FLNB, and DAMM1 were shown to distribute to the organ of Corti and hepatocyte plasma membrane. In the mouse liver, CX26 and TJP1 co-localized at the plasma membrane. In conclusion, CX26 associates with components of other membrane junctions that integrate with the cytoskeleton.

TGF‐β1 and connexin‐43 expression in neurogenic bladder from rats with sacral spinal cord injury

Sacral spinal cord injury (SCI) could induce underactive bladder (UAB). Malfunction of connexin 43 (CX43) regulated by TGF-β1 might involve in urinary bladder dysfunction. We studied the changes of CX43 and TGF-β1/Smad3 signaling in detrusor of neurogenic bladder (NB) in sacral SCI rats. Sacral SCI was produced by hemisection (SSCH) or transection (SSCT) of spinal cord between L4 and L5 in female Wistar rats. BBB scores, residual urine volume and bladder weight as well as characteristic cystometric parameters at 6th week were used to confirm the successful establishment of NB. Western blotting and qRT-PCR were used to exam the protein and mRNA expression levels of CX43, CX45, TGF-β1, and Smad3 in detrusor. BBB scores were significantly decreased, with the lowest in SSCT rats (P < 0.01). The residual urine volume, mean bladder weight, and cystometric parameters were increased, with the highest in SSCT rats. CX43 and phospho-CX43 protein levels were significantly decreased, but those of TGF-β1, Smad3, and phospho-Smad3 were significantly increased. It was the protein and mRNA levels of CX43 but not those of CX45 which were decreased in negative accordance with those of TGF-β1 and Smad3. Those changes were more significant in SSCT than in SSCH rats. This study indicates that voiding dysfunction is related to the decreased CX43 function in detrusor from NB. TGF-β1/Smad3 signaling might be involved in the down-regulation of CX43 in SCI rats. Early regulation of CX43 might be beneficial to patients with voiding dysfunction.

Effects of gap junctions blockers on fast ripples and connexin in rat hippocampi after status epilepticus

ObjectiveTo assess the dynamic changes in the average and peak spectral power of fast ripples (FRs) in the hippocampi after interventions with valproate sodium (VPA), carbenoxolone (CBX) and quinine (QUIN). MethodAdult rats were used to establish a lithium–pilocarpine (pilo) epileptic model, and were assigned to a lithium–pilocarpine (PILO), VPA + PILO, QUIN + PILO or CBX + PILO group. Intracranial electroencephalography was performed before and after status epilepticus (SE). The hippocampal connexin (CX) 43, CX32 and CX36 expressions were analyzed via western blotting. ResultsThe time required for the disappearance of SE after chloral hydrate injection was lower in the intervention groups than in the PILO group (p < 0.05). Seizures induced CX43 expression, but had no significant effects on CX36 or CX32 expressions. Pretreatment with VPA, QUIN and CBX inhibited CX43, CX36 and CX32 expression after SE. The average spectral power of the FRs was significantly lower in the VPA + PILO and QUIN + PILO groups than in the PILO group at 10 min after SE, 10 min before chloral hydrate injection, and 10 min after chloral hydrate injection (p < 0.05). The average spectral power of FRs was lower in the CBX + PILO group than in the PILO group at 10 min after SE (p < 0.05). The average spectral power of FRs in the 3 intervention groups recovered to the baseline level at 10 min after chloral hydrate injection and persisted for 3 days after SE. The dynamic changes in the average and peak spectral power of FRs were similar. SignificanceAfter SE, CX may participate in pathological FR generation by establishing abnormal electrical synaptic transmission. Gap junction blockers can inhibit various CX expressions, and thus decrease FR energy and alleviate the degree of seizure. These findings could contribute to the development of new anti-epileptic drugs with novel mechanistic targets.

The impact of connexins on atherogenesis: A brief review

Connexins family in humans consists of 21 highly conserved proteins that are responsible for contact formation between cells. On the cell surface, connexins form hemichannels, or connexons. Two hemichannels brought together form a gap junction, a form of intercellular contact that allows for direct transfer of material and signals between the adjacent cells. Gap junctions serve for transporting ions and other soluble, low molecular weight molecules therefore synchronizing the microenvironment of the contacting cells, and maintaining cell and tissue homeostasis. Impairment of gap junctions is associated with different pathological conditions. Importantly, it has been described in atherosclerosis, which causes local cellular dysfunction in the arterial wall tissues followed by the development of atherosclerotic plaque. There are 3 main connexins expressed in human cardiovascular system: Connexin 37 (Cx37), Cx40, and Cx43. Alterations in the arterial wall cells observed in atherosclerosis include changes in the expression pattern of the main connexins, and impairment of intercellular contacts and communication. According to the currently available data, Cx37 and Cx40 have anti-atherogenic and vasculoprotective properties, while Cx43 appears to be more pro-atherogenic. However, the effects of connexins are cell type-dependent and in many cases, remain to be studied in detail. In this review, we summarize the available knowledge on connexins of the arterial wall cells involved in atherosclerosis development.

Localization and distribution of connexin 43 in mitochondria of cervical cancer HeLa cells

Objective To study the distribution of connexin 43 (Cx43) in cervical cancer HeLa cells, and to verify the localization of Cx43 in mitochondria. Methods HeLa cells were segregated into cytoplasm, cell nucleus, mitochondria and supernatant after segregation by using the method of homogenate and centrifuge. Immunoelectron microscope was used to observe the morphology of mitochondria and the localization as well as the distribution of Cx43 in HeLa cells. Voltage-dependent anion channel 1 (VDAC1) was used to confirm the localization of mitochondria. Immunofluorescence was used to costain HeLa cells with Cx43 and mitochondrial marker VDAC1 to verify mitochondria localization of Cx43 in cervical cancer HeLa cells. Then Western blot was used to quantify the expression of Cx43 in fractions (cytoplasmic fraction, nuclear, mitochondria and post mitochondrial supernatant). Mitochondrial markers including VDAC1 and cytochrome c oxidase Ⅳ (COX Ⅳ) were used to confirm mitochondria. Plasma membrane marker (LHR) was used to confirm plasma membrane. Results Immunoelectron microscope confirmed that the normal mitochondria or cystic swollen one could be seen in the complete HeLa cells and the detached HeLa cells mitochondria, with the presence of Cx43 and VDAC1 in detached mitochondria. Immunofluorescence showed Cx43 colocalized with VDAC1. There was a significant difference in the Cx43 expressions of the subcellular structure in the HeLa cells [cytoplasm (1.23±0.11), cell nucleus (0.39±0.09), mitochondria (3.67±0.59), supernatant after segregation (0.16±0.06); F = 84.17, P < 0.05]. It also showed that the relative amount of Cx43 in mitochondria was enriched. Conclusions Cx43 is enriched in mitochondria in cervical cancer HeLa cells. Therefore, Cx43 in mitochondria might be a potential target in diagnosis, therapy and prognosis of cervical cancer. Key words: HeLa cells; Connexin 43; Mitochondria; Localization; Distribution

Effects of Di ao Xin xue Kang on connexin 43 of myocardial gap junction after acute ischemia injury

Objective To observe the effects of Di ao Xin xue Kang on connexin 43(Cx43), in order to make clear the protective mechanism against ischemia-induced arrhythmias. Methods The rats were randomly divided into 3 groups according to the digital table: SM operation group(group SM, n=10), ischemia group(group IM, n=10), Di ao Xin xue Kang group(group DA, n=10). Myocardial infarction was induced by legation the left anterior descending coronary artery for 120 min.The expression of Cx43 of the ischemic myocardium was studied by immunohistochemistry technique.The ventricular arrhythmia was observed, and MDA, SOD levels were measured. Results Compared with the SM group, the arrhythmia score[(3.8±0.7)points vs.(0.7±0.2)points, t=13.466, P<0.01]and serum MDA level[(8.3±0.8)nmol/mL vs.(4.2±0.7)nmol/mL, t=12.197, P<0.01]in the IM group were significantly increased, SOD activity in the IM group[(68.2±5.5)U/mL vs.(144.1±7.9)U/mL, t=24.932, P<0.01]decreased, and serum Cx43 content[(0.39±0.06) vs.(0.92±0.05), t=21.459, P<0.01]significantly decreased.Compared with IM group, the arrhythmia score of the DA group[(2.6±0.8) vs.(3.8±0.7), t=3.570, P<0.01]decreased, and serum Cx43 content[(0.60 ±0.08) vs.(0.39±0.06), t=6.641, P<0.01]increased and distributed regularly. Conclusion Di ao Xin xue Kang can effectively alleviate Cx43 degrading induced by acute myocardial infarction, the mechanism of this effect is associated with resisting oxidative stress. Key words: Myocardial ischemia; Arrhythmias, cardiac; Ischemic preconditioning, myocardial; Connexin 43; Di Ao Xin Xue Kang

Involvement of mitochondrial mechanisms in myocardial protection by ischemic postconditioning

Background Myocardial ischemia/reperfusion injury (MI/RI) is life-threaten to the patients with cardiovascular diseases, and is a serious risk factor for the mortality of patients with diabetes mellitus. Ischemic postconditioning (IPO) effectively protects myocardia probably by strengthening the function of mitochondria. Objective To summarize mitochondrial mechanisms underlying myocardial protection after IPO. Content IPO protects myocardia probably by activating endogenous signal transduction pathways, involved in reperfusion injury salvage kinase(RISK) and survival activation factor enhancement(SAFE), followed by the release of endogenous trigger factors, such as adenosine. Adenosine can activate signal transduction and activator of transcription 3 (STAT3) and protein kinase C (PKC), then promote the phosphorylation of glycogen synthase kinase-3β (GSK-3β) , open mitochondrial sensitive potassium (mitoKATP) channel, and shut off mitochondrial permeability transition pores (mPTP) by increasing the expression of mitochondrial cardiolipin, connexin 43 (Cx43) and anti-apoptosis gene Bcl-2, thereafter remaining thioredoxin (Trx)2 and mitochondrial respiratory chain intact, and reduce MI/RI. Trend IPO protects myocardia in patients with diabetes mellitus by maintaining homeostasis of mitochondria. However, further investigations are needed to validate the proposed underlying mechanisms of myocardial protection by IPO, especially in diabetic MI/RI, and to confirm some specific signal transduction pathways as new therapeutic targets for MI/RI. Key words: Ischemia-reperfusion injury; Myocardial; Postconditioning; Mitochondria; Myocardial protection

Connexin 43 coupling in bovine cumulus cells, during the follicular growth phase, and its relationship to in vitro embryo outcomes.

Gap junctional coupling between cumulus cells is required for oocytes to reach developmental competence. Multiple connexins, which form these gap junctions, have been found within the ovarian follicles of several species including bovine. The aim of this study was to determine the role of connexin 43 (CX43) and its relationship to embryo development, after in vitro fertilization (IVF). Cumulus-oocyte complexes (COCs) were obtained from abattoir sourced, mixed breed, bovine ovaries. COCs were isolated from follicles ranging from 2 to 5 mm in size, representing the preselected follicle pool. Immediately after isolation, two cumulus cell biopsies were collected and stored for analysis pending determination of developmental outcomes. Using in vitro procedures, COCs were individually matured, fertilized, and cultured to the blastocyst stage. Biopsies were grouped as originating from COCs that arrested at the two-cell stage (low developmental competence [LDC]) or having developed to the late morula/blastocyst stage (high developmental competence [HDC]), after IVF and embryo culture. The expression level of CX43 was found to be significantly higher in cumulus cells from COCs that had an HDC when compared with those that had an LDC. Moreover, the gap junctional intercellular coupling rate was significantly higher in cumulus from COCs deemed to have an HDC. Significantly higher expression of the cumulus health markers luteinizing hormone receptor and cytochrome p450 19A1 was found in the cumulus originating from oocytes with HDC, suggesting that this system may provide a mechanism for noninvasively testing for oocyte health in preselected bovine follicles.

Connexin43 and Myocardial Ischemia-Reperfusion Injury.

Recently, the treatment and prevention of ischemic cardiomyopathy is one of the emerging research topics in the cardiovascular field. Gap junction is the basic structure of cardiac electrophysiology. Connexin is the basic unit of gap junctions. Connexin43(CX43) is the most abundant member of Cx family in the heart, the normal expression of Cx43 is important for heart development, electrically coupled cardiomyocytes activities and coordination of myocardial function. The connection between Cx43 and myocardial ischemia/reperfusion or reperfusion injury has become the focus of current research. We undertook a structured search of bibliographic database for peer-reviewed research literature using a focused review question and inclusion/exclusion criteria. The quality of retrieved papers was appraised using standard tools. The characteristics of screened papers were described, and a deductive qualitative content analysis methodology was applied to analyze the interventions and findings of included studies using a conceptual framework. Twenty-one papers were included in the review, eight papers outlined the relationship of Cx43 and reperfusion arrhythmias. Eight papers pointed out the effect on the infarct size of Cx43. The findings of this review confirm that Cx43 is the most abundant member of Cx family in the heart and is vital for myocardial protection during ischemia/reperfusion process and for ischemia/reperfusion injury. Many of its mechanism are still not very clear and require future research in the future.

Angiopoietin-2 regulates vascular hyporeactivity through myoendothelial gap junction

Objective To observe the mediation role of myoendothelial gap junction (MEGJ) in the regulation of angiopoietin-2 (Ang2) on vascular hyporeactivity after hypoxia, and figure out the responsible Cx. Methods The double-sided cells co-culture model of vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) were set up, the VSMCs contractile was detected via the leakage of fluoresceine isothiocyanate-bovine serum albumin (FITC-BSA), MEGJ formation was observed by immunocytochemistry of phalloidin, and MEGJ communication was reflected by the Sulforhodamine B transfer. Results (1) In the double-sided cells co-culture model, protein expression of iNOS after hypoxia increased more in VSMCs than that in VECs (3.6 times, P=0.003), and the increased iNOS expression in VSMCs could be decreased by Ang2 siRNA (by 60%, P=0.003). (2) The MEGJ non-specific inhibitor 18α-GA, the specific Cx43 siRNA, or angiopoietin receptor 2 (Tie2) siRNA could suppress the iNOS expression in VSMCs (P=0.001, 0.001, and 0.001, respectively), and improved the VSMCs contractile reactivity after hypoxia following Ang2 treatment (P=0.004, 0.009, and 0.001, respectively). (3) Cx43 siRNA or Tie2 siRNA could inhibit the increased MEGJ formation (P=0.005 and 0.005, respectively), and inhibit the increased MEGJ communication after hypoxia following Ang2 treatment (P=0.003 and 0.004, respectively). Conclusion MEGJ mediated VECs-dependent regulation of Ang2 on vascular hyporeactivity after hypoxia, Cx43 was the responsible Cx isoform. Key words: Hypoxia; Vascular hyporeactivity; Angiopoietin-2; Myoendothelial gap junction; Connexin 43

CX43 is essential for optimal cGAS function during cytosolic DNA-sensing

Abstract During intracellular Chlamydia infection, cytosolic DNA is sensed by cyclic GMP-AMP synthase (cGAS) that catalyzes the synthesis of cGAMP from ATP and GTP. cGAMP is a key mediator of IFN beta expression during DNA sensing and can also transfer to adjacent uninfected cells through intercellular connexin gap-junctions to induce intrinsic immunity. In this study, we investigated the role of gap junction protein connexin 43 (CX43) during Chlamydia infection. Using RNA-in situ hybridization, we discovered a distinct intracellular contribution of CX43 in IFN beta expression in the infected cells and cells transfected with DNA, a function distinct from its intercellular gap-junction role affecting IFN beta expression in adjacent uninfected cell. CX43 is essential for IFN beta expression during DNA-sensing upstream of STING activation, but not required during RNA-sensing. CX43 co-localized with transfected DNA and on the chlamydial inclusion membrane with cGAS. CX43-depleted cells showed significantly reduced cGAMP production during DNA-sensing. CX43 forms hemichannels on intracellular membrane, which open and close to allow nucleotide transfer. Blocking this transport across CX43 hemichannel reduced IFN beta expression during infection and DNA-sensing, without altering CX43 localization. These results uncover a novel role of CX43 in cGAMP synthesis by cGAS during cytosolic DNA-sensing.

Effects of Fullerenol Nanoparticles on Rat Oocyte Meiosis Resumption.

The excellent biocompatibility and biological effects of fullerenol and its derivatives make their biomedical application promising. The potential effects of fullerenol in mammals have been extensively studied, but little is known about its effects on female reproduction. Using canonical oocyte-granulosa cell complexes (OGCs) in vitro maturation culture model, we investigated the effect of fullerenol on the first oocyte meiotic resumption. In the surrounding granulosa cells, fullerenol nanoparticles occluded the extracellular domain of the epidermal growth factor receptor (EGFR) to reduce EGFR-ligand binding and subsequent extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation, which involved the regulation of connexin 43 (CX43) expression and internalization. Downregulation of CX43 expression and the retraction of transzonal projections (TZPs) interrupted the gap junction channel and TZPs based mass transportation. This effect decreased cyclic adenosine monophosphate (cAMP) levels in the oocyte and thereby accelerated rat oocyte meiosis resumption. Moreover, perinuclear distribution of CX43 and EGFR was observed in granulosa cells, which could further exacerbate the effects. Fullerenol nanoparticles interfered with the strict process of oocyte meiosis resumption, which likely reduced the oocyte quality.

Engraftment of Human Pluripotent Stem Cell-derived Progenitors in the Inner Ear of Prenatal Mice

There is, at present, no curative treatment for genetic hearing loss. We have previously reported that transuterine gene transfer of wild type CONNEXIN30 (CX30) genes into otocysts in CX30-deleted mice could restore hearing. Cell transplantation therapy might be another therapeutic option, although it is still unknown whether stem cell-derived progenitor cells could migrate into mouse otocysts. Here, we show successful cell transplantation of progenitors of outer sulcus cell-like cells derived from human-derived induced pluripotent stem cells into mouse otocysts on embryonic day 11.5. The delivered cells engrafted more frequently in the non-sensory region in the inner ear of CX30-deleted mice than in wild type mice and survived for up to 1 week after transplantation. Some of the engrafted cells expressed CX30 proteins in the non-sensory region. This is the first report that demonstrates successful engraftment of exogenous cells in prenatal developing otocysts in mice. Future studies using this mouse otocystic injection model in vivo will provide further clues for developing treatment modalities for congenital hearing loss in humans.

Connexin 43 and tumors

Cell gap junction is a special protein channel. Gap junction-mediated exchange of information between cells is crucial for cell growth, differentiation and tissue homeostasis. Connexin 43 (Cx43) is one of the members of the gap junction protein family. In recent years, researches show that abnormal Cx43 gene expression leads to the cell gap junctional communication dysfunction, which is closely related to the occurrence, metastasis and prognosis of a variety of tumors. Cx43 is expected to become a new target for clinical diagnosis and treatment of tumors. Key words: Connexin 43; Neoplasms; Cell communication