Gap Junction Gene Research Articles

VEGF-dependent testicular vascularisation involves MEK1/2 signalling and the essential angiogenesis factors, SOX7 and SOX17

BackgroundAbnormalities of in utero testis development are strongly associated with reproductive health conditions, including male infertility and testis cancer. In mouse testes, SOX9 and FGF9 support Sertoli cell development, while VEGF signalling is essential for the establishment of vasculature. The mitogen-activated protein kinase (MAPK) pathway is a major signalling cascade, essential for cell proliferation, differentiation and activation of Sry during primary sex-determination, but little is known about its function during fetal testis morphogenesis. We explored potential functions of MAPK signalling immediately after the establishment of testis cords in embryonic day (E)12.5 Oct4-eGFP transgenic mouse testes cultured using a MEK1/2 inhibitor.ResultsRNA sequencing in isolated gonadal somatic cells identified 116 and 114 differentially expressed genes after 24 and 72 h of MEK1/2 inhibition, respectively. Ingenuity Pathway Analysis revealed an association of MEK1/2 signalling with biological functions such as angiogenesis, vasculogenesis and cell migration. This included a failure to upregulate the master transcriptional regulators of vascular development, Sox7 and Sox17, VEGF receptor genes, the cell adhesion factor gene Cd31 and a range of other endothelial cell markers such as Cdh5 (encoding VE-cadherin) and gap junction genes Gja4 and Gja5. In contrast, only a small number of Sertoli cell enriched genes were affected. Immunofluorescent analyses of control testes revealed that the MEK1/2 downstream target, ERK1/2 was phosphorylated in endothelial cells and Sertoli cells. Inhibition of MEK1/2 eliminated pERK1/2 in fetal testes, and CD31, VE-cadherin, SOX7 and SOX17 and endothelial cells were lost. Consistent with a role for VEGF in driving endothelial cell development in the testis, inhibition of VEGFR also abrogated pERK1/2 and SOX7 and SOX17 expressing endothelial cells. Moreover, while Sertoli cell proliferation and localisation to the testis cord basement membrane was disrupted by inhibition of MEK1/2, it was unaffected by VEGFR inhibition. Instead, inhibition of FGF signalling compromised Sertoli cell proliferation and localisation to the testis cord basement membrane.ConclusionsTogether, our data highlight an essential role for VEGF-dependent MEK1/2 signalling in promoting vasculature and indicate that FGF signalling through MEK1/2 regulates Sertoli cell organisation in the developing mouse testis.

Chronic dietary exposure to glyphosate-induced connexin 43 autophagic degradation contributes to blood-testis barrier disruption in roosters

Glyphosate (GLY) is the most universally used herbicide worldwide and its application has caused extensive pollution to the ecological environment. Increasing evidence has revealed the multi-organ toxicity of GLY in different species, but its male reproductive toxicity in avian species remains unknown. Thus, in vivo and in vitro studies were conducted to clarify this issue. Data firstly showed that chronic GLY exposure caused testicular pathological damage. Intriguingly, we identified and verified a marked down-regulation gap junction gene Connexin 43 (Cx43) in GLY-exposed rooster testis by transcriptome analysis. Cx43 generated by Sertoli cells acts as a key component of blood-testis barrier (BTB). To further investigate the cause of GLY-induced downregulation of Cx43 to disrupt BTB, we found that autophagy activation is revealed in GLY-exposed rooster testis and primary avian Sertoli cells. Moreover, GLY-induced Cx43 downregulation was significantly alleviated by ATG5 knockdown or CQ administration, respectively, demonstrating that GLY-induced autophagy activation contributed to Cx43 degradation. Mechanistically, GLY-induced autophagy activation and resultant Cx43 degradation was due to its direct interaction with ER-α. In summary, these findings demonstrate that chronic GLY exposure activates autophagy to induce Cx43 degradation, which causes BTB damage and resultant reproductive toxicity in roosters.

Hepatotoxicity of cyanotoxin microcystin-LR in human: Insights into mechanisms of action in the 3D culture model Hepoid-HepaRG

Microcystin-LR (MC-LR) is a potent hepatotoxin produced by harmful cyanobacterial blooms (CyanoHABs). MC-LR targets highly differentiated hepatocytes expressing organic anion transporting polypeptides OATP1B1 and OATP1B3 that are responsible for hepatocellular uptake of the toxin. The present study utilized an advanced 3D in vitro human liver model Hepoid-HepaRG based on the cultivation of collagen-matrix embedded multicellular spheroids composed of highly differentiated and polarized hepatocyte-like cells. 14-d-old Hepoid-HepaRG cultures showed increased expression of OATP1B1/1B3 and sensitivity to MC-LR cytotoxicity at concentrations >10 nM (48 h exposure, EC20 = 26 nM). MC-LR induced neither caspase 3/7 activity nor expression of the endoplasmic reticulum stress marker gene BiP/GRP78, but increased release of pro-inflammatory cytokine IL-8, indicating a necrotic type of cell death. Subcytotoxic (10 nM) and cytotoxic (≥100 nM) MC-LR concentrations disrupted hepatocyte functions, such as xenobiotic metabolism phase-I enzyme activities (cytochrome P450 1A/1B) and albumin secretion, along with reduced expression of CYP1A2 and ALB genes. MC-LR also decreased expression of HNF4A gene, a critical regulator of hepatocyte differentiation and function. Genes encoding hepatobiliary membrane transporters (OATP1B1, BSEP, NTCP), hepatocyte gap junctional gene connexin 32 and the epithelial cell marker E-cadherin were also downregulated. Simultaneous upregulation of connexin 43 gene, primarily expressed by liver progenitor and non-parenchymal cells, indicated a disruption of tissue homeostasis. This was associated with a shift in the expression ratio of E-cadherin to N-cadherin towards the mesenchymal cell marker, a process linked to epithelial-mesenchymal transition (EMT) and hepatocarcinogenesis. The effects observed in the human liver cell in vitro model revealed mechanisms that can potentially contribute to the MC-LR-induced promotion and progression of hepatocellular carcinoma (HCC). Hepoid-HepaRG cultures provide a robust, accessible and versatile in vitro model, capable of sensitively detecting hepatotoxic effects at toxicologically relevant concentrations, allowing for assessing hepatotoxicity mechanisms, human health hazards and impacts of environmental hepatotoxins, such as MC-LR.

Cx26 heterozygous mutations cause hyperacusis-like hearing oversensitivity and increase susceptibility to noise.

Gap junction gene GJB2 (Cx26) mutations cause >50% of nonsyndromic hearing loss. Its recessive hetero-mutation carriers, who have no deafness, occupy ~10 to 20% of the general population. Here, we report an unexpected finding that these heterozygote carriers have hearing oversensitivity, and active cochlear amplification increased. Mouse models show that Cx26 hetero-deletion reduced endocochlear potential generation in the cochlear lateral wall and caused outer hair cell electromotor protein prestin compensatively up-regulated to increase active cochlear amplification and hearing sensitivity. The increase of active cochlear amplification also increased sensitivity to noise; exposure to daily-level noise could cause Cx26+/- mice permanent hearing threshold shift, leading to hearing loss. This study demonstrates that Cx26 recessive heterozygous mutations are not "harmless" for hearing as previously considered and can cause hyperacusis-like hearing oversensitivity. The data also indicate that GJB2 hetero-mutation carriers are vulnerable to noise and should avoid noise exposure in daily life.

Evaluating gap junction variants for a role in pediatric cataract: an overview of the genetic landscape and clinical classification of variants in the GJA3 and GJA8 genes

ABSTRACT Introduction Variants in the two lens-expressed gap junction genes GJA3 and GJA8 are among the most common causes of inherited pediatric cataract. These two genes alone account for up to 18% of the cases, second only to the crystallin gene family. Areas covered All published cataract-associated variants in the GJA3 and GJA8 genes were reviewed for a role in pediatric cataract. Autosomal dominant inheritance was most frequently reported, alongside instances of reduced penetrance and autosomal recessive disease. Variant curation using the ACMG-AMP guidelines identified that many variants do not meet the modern standards for clinical interpretation of pathogenicity. There is broad phenotypic heterogeneity of cataract associated with gap junction gene variants. Pathogenic variants are located throughout both proteins with an enrichment in the N-terminal, first two transmembrane domains, and two extracellular loops. Expert opinion Nearly half the gap junction gene variants observed in cataract patients lack sufficient evidence of pathogenicity to form a useful clinical opinion. For many variants, this may be rectified over time as the variant is observed in additional patients but would be vastly accelerated by the generation of well-characterized and standardized functional data evaluating the specific effect of each variant on protein function.

Genome-wide identification of gap junction (connexins and pannexins) genes in black rockfish (Sebastes schlegelii): Evolution and immune response mechanism following challenge

Cell-to-cell communication through gap junction channels is very important to coordinate the functions of cells in all multicellular biological tissues. It allows the direct exchange of ions and small molecules (including second messengers, such as Ca2+, IP3, cyclic nucleotides, and oligonucleotides). In this study, a total of 48 members of the gap junction (GJ) protein family were identified from Sebastes schlegelii. In S. schlegelii, GJ proteins were classified into two types, connexin, and pannexin, and then connexins were divided into five subfamilies. The naming of 48 genes was verified through phylogenetic analysis and syntenic analysis. The connexin proteins contained four transmembrane fragments and two extracellular loops, the lengths of the intracellular loop and C-terminal was quite different, and the C-terminal region was highly variable after post-translational modification. PPI analysis showed that GJs interacted with tight junctions, adhesive junctions, and cell adhesions to form a complex network and participated in cell-cell junction organization, ATP binding, ion channel, voltage-gated conduction, wnt signaling pathway, Fc-γ receptor signaling pathway, and DNA replication. In addition, the S. schlegelii GJ protein was highly expressed in intestinal tissues and remarkably regulated after Edwardsiella tarda and Streptococcus iniae infection. The expression of GJs in intestinal cells of S. schlegelii was significantly regulated by LPS and poly (I:C), which was consistent with the results of intestinal tissue stimulation by pathogens. In conclusion, this study can provide valuable information for further research on the function of S. schlegelii GJ proteins.

Tanshinone IIA enhances the ovarian reserve and attenuates ovarian oxidative stress in aged mice.

BackgroundTanshinone IIA (TSA), a major lipophilic component extracted from the roots of Salvia miltiorrhiza Bunge, has been widely used in China for its various biological activities. However, its effect on ovarian reserve in aged mice was not studied elsewhere.ObjectivesThis study aimed to explore the effect of TSA on the ovarian reserve of aged mice as well as young mice. Forty weeks old mice (N = 40) were considered as aged group compared to 4 weeks old mice (N = 40), and these groups were subdivided into four subgroups (N = 10) to receive different doses of TSA (0, 10, 20, and 40 μg/g/day).MethodsThe effect of TSA was evaluated by counting follicular number by histological examination. Basal serum levels of FSH, LH, E2, and anti‐Mullerian hormone (AMH) were measured by ELISA. Moreover, the expression levels of antioxidant genes (CAT, Nrf2, GPX1), gap junction (Cx37), ERK1/2, and Smad5 family gene were examined at both mRNA (qPCR) and protein levels (western blot).ResultsFollicular number, level of AMH and E2, and the expression of CAT, Nrf2, and GPX1 genes increased significantly (p < 0.05) in aged mice administrated with medium (20 μg/g/day) and high (40 μg/g/day) doses of TSA, whereas FSH and LH levels were significantly low compared to low dose (10 μg/g/day) and control (0 μg/g/day) aged subgroups. However, we did not observe any effect of all doses of TSA on young mice.ConclusionsAdministration of TSA with medium and high doses up‐regulates the expression of antioxidative genes, reduces the oxidative injury, increases levels of AMH, and E2 levels that are relatively comparable to those in young mice, and consequently results in a healthy oocyte development.

Connexin Mutations and Hereditary Diseases.

Inherited diseases caused by connexin mutations are found in multiple organs and include hereditary deafness, congenital cataract, congenital heart diseases, hereditary skin diseases, and X-linked Charcot–Marie–Tooth disease (CMT1X). A large number of knockout and knock-in animal models have been used to study the pathology and pathogenesis of diseases of different organs. Because the structures of different connexins are highly homologous and the functions of gap junctions formed by these connexins are similar, connexin-related hereditary diseases may share the same pathogenic mechanism. Here, we analyze the similarities and differences of the pathology and pathogenesis in animal models and find that connexin mutations in gap junction genes expressed in the ear, eye, heart, skin, and peripheral nerves can affect cellular proliferation and differentiation of corresponding organs. Additionally, some dominant mutations (e.g., Cx43 p.Gly60Ser, Cx32 p.Arg75Trp, Cx32 p.Asn175Asp, and Cx32 p.Arg142Trp) are identified as gain-of-function variants in vivo, which may play a vital role in the onset of dominant inherited diseases. Specifically, patients with these dominant mutations receive no benefits from gene therapy. Finally, the complete loss of gap junctional function or altered channel function including permeability (ions, adenosine triphosphate (ATP), Inositol 1,4,5-trisphosphate (IP3), Ca2+, glucose, miRNA) and electric activity are also identified in vivo or in vitro.

Cx43 upregulation in HUVECs under stretch via TGF-β1 and cytoskeletal network.

Many physiological and pathophysiological processes in cells or tissues are involved in mechanical stretch, which induces the gap junction gene expression and cytokine TGF beta changes. However, the underlying mechanisms of the gap junction gene expression remain unknown. Here, we showed that the mRNA and protein levels of Cx43 in human umbilical vein endothelial cells (HUVECs) were significantly increased after 24 h stretch stimulation, and TGF beta1 (not TGF beta2) expression was also upregulated. Administration of TGF beta1 into HUVECs without stretch also induced upregulation of Cx43 expression. However, SB431542, a specific inhibitor of the TGF beta1 receptor, blocked the Cx43 protein upregulation caused by TGF beta1. Further, the increase of Cx43 protein expression under the stretch condition was partially blocked by SB431542; it was also partially blocked by simultaneous administration of anti-TGF beta1 monoclonal neutralization antibody. Importantly, the upregulation of Cx43 induced by stretch was blocked by the administration of actin and microtubule inhibitors, while NEDD4, a key element in mediating Cx43 protein ubiquitination and degradation, was not changed under the stretch condition. In conclusion, upregulation of Cx43 expression under the 24 h stretch condition is mediated via TGF beta1 receptor signaling pathway, and it also involves the actin and microtubule cytoskeletal network.

BOEC-Exo Addition Promotes In Vitro Maturation of Bovine Oocyte and Enhances the Developmental Competence of Early Embryos.

Simple SummaryThe results of the present study proved that the addition of bovine oviductal epithelial cell derived exosomes (BOEC–Exo) to the in vitro maturation (IVM) media improved the bovine oocyte maturation and early embryo development. The addition of BOEC–Exo not only significantly enhanced the polar body exclusion, but also enhanced the expression of connexins in cumulus oocyte complexes (COCs). Likewise, the reactive oxygen species (ROS) level, protein expressions of SIRT-1, and mitochondrial membrane potential (ΔΨm) also suggested that BOEC–Exo addition to IVM media is highly beneficial for in vitro bovine oocyte maturation. Furthermore, BOEC–Exo treatment to the primary cultured bovine cumulus cells significantly attenuated apoptosis, which also showed its positive influence on the COCs. Moreover, oocytes that were matured in the presence of BOEC–Exo led to the production of a significantly higher quantity and quality of day-8 blastocysts. Additionally, the BOEC–Exo treated blastocysts had a higher implantation potential when compared with the control. Our results suggest that the addition of BOEC–Exo to IVM media significantly enhanced the percentage of oocytes maturation and improved the embryo quantity and quality. Exosomes are nano-sized vesicles with abundant nucleic acids, proteins, lipids, and other regulatory molecules. The aim of this study was to examine the effect of BOEC–Exo on bovine in vitro oocyte maturation and in vitro embryo development. We found that a 3% Exo supplementation to IVM media significantly enhanced the oocyte maturation and reduced the accumulation of ROS in MII-stage bovine oocytes. Oocyte maturation related genes (GDF9 and CPEB1) also confirmed that 3% Exo treatment to oocytes significantly (p < 0.05) enhanced the oocyte maturation. Next, we cultured bovine cumulus cells and assessed the effects of 3% Exo, which showed a reduced level of apoptotic proteins (caspase-3 and p-NF-κB protein expressions). Furthermore, we examined the gap junction (CX43 and CX37) and cumulus cells expansion related genes (HAS2, PTX3, and GREM1) in cumulus–oocyte complexes (COCs), and all those genes showed significantly (p < 0.05) higher expressions in 3% Exo-treated COCs as compared with the control group. Moreover, peroxisome proliferator-activated receptors (PPARs) and lipid metabolism-related genes (CPT1 and FABP3) were also analyzed in both the control and 3% Exo groups and the results showed significant (p < 0.05) enhancement in the lipid metabolism. Finally, the oocytes matured in the presence of 3% Exo showed a significantly higher rate of embryo development and better implantation potential. Finally, we concluded that Exo positively influenced bovine oocyte in vitro maturation and improved the early embryo’s developmental competence.

Genome-wide characterization of gap junction (connexins and pannexins) genes in turbot (Scophthalmus maximus L.): evolution and immune response following Vibrio anguillarum infection.

Gap junction (GJ), a special intercellular junction between different cell types, directly connects the cytoplasm of adjacent cells, allows various molecules, ions and electrical impulses to pass through the intercellular regulatory gate, and plays vital roles in response to bacterial infection. Up to date, the information about the GJ in turbot (Scophthalmus maximus L.) is still limited. In current study, 43 gap junction genes were identified in turbot, phylogeny analysis suggested that gap junctions from turbot and other species were clustered into six groups, GJA, GJB, GJC, GJD, GJE and PANX, and turbot GJs together with respective GJs from Japanese flounder, half-smooth tongue sole and large yellow croaker, sharing same ancestors. In addition, these 43 GJ genes distributed in different chromosomes unevenly. According to gene structure and domain analysis, these genes (in GJA-GJE group) were highly conserved in that most of them contain the transmembrane area, connexin domain (CNX) and cysteine-rich domain (connexin CCC), while PANXs contain Pfam Innexin. Although only one tandem duplication was identified in turbot gap junction gene, 235 pairs of segmental duplications were identified in the turbot genome. To further investigate their evolutionary relationships, Ka/Ks was calculated, and results showed that most ratios were lower than 1, indicating they had undergone negative selection. Finally, expression analysis showed that gap junction genes were widely distributed in turbot tissues and significantly regulated after Vibrio anguillarum infection. Taken together, our research could provide valuable information for further exploration of the function of gap junction genes in teleost.

Post-thawing and culture comparison of three routine slow freezing methods for human ovarian tissue cryopreservation: Histological, molecular, and hormonal aspects

To find the gold standard out of three pre-established routine slow freezing (SF) methods, ovarian cortex tissues of nine transsexual individuals were cryopreserved and compared to each other, as well as the control (fresh) samples. Histological, genomic, and endocrinological effects of the SFs were assessed post-thawing and after a seven-day culture period. SF1 included 10% dimethyl-sulfoxide (Me2SO) in the base medium (BM), SF2 had 1.5M/L ethylene-glycol (EG) and 0.1M/L sucrose in the BM, and SF3 consisted of 6% Me2SO, 6% EG and 0.15M/L sucrose in the BM. The cortical tissue strips went under a programmed cooling process and were stored in liquid nitrogen. Histological criteria (tissue damage and follicular quality), as well as gene expression levels, were assessed in the thawed and control tissues. Half of the thawed and control tissues were cultured for seven days and their histology, genetic profile, and hormonal status were examined as the reflection of the avascular tension effect. Post-thawing tissue damage was similar between all groups but significantly increased post-culture (P<0.05). The percentages of high-quality follicles diminished in all SFs after thawing and culture (P<0.05) except for the similarity of post-thawing SF3, compared to control. The genetic profile of the tissue after thawing and culture suggested quiescence/activation balance in SF1 and 2 and significant down-regulation in SF3, compared to the control specimens (P<0.05). Post-thawing BAX:BCL2 was higher than control in SF1 and SF3 (P<0.05), while this ratio in SF2 was similar to the control. However, after culture this ratio was similar to that of control in SF3 and diminished in SF1 and 2 (P<0.05). The expression levels of gap-junction genes showed dramatic pre- and post-thawing fluctuations in all groups. After culture, estradiol in SF3 was significantly higher than SF1 and 2 (P<0.05). In addition, progesterone in SF3 was similar to control but significantly lower in SF1 and 2 (P<0.05). In conclusion, all SFs showed advantages and disadvantages, and the follicular quality and its function depend on the type of cryoprotectant and the speed of thawing. The effects of freezing/thawing continue to appear during the seven days of culture. According to the results of this study, SF3 seems to be more promising in keeping the follicles functional and safe from cell damage during culture.

Single-cell RNA-sequencing analysis of the ciliary epithelium and contiguous tissues in the mouse eye

The ciliary epithelium plays a central role in ocular homeostasis but cells of the pigmented and non-pigmented layers are difficult to isolate physically and study. Here we used single-cell RNA-sequencing (scRNA-seq) to analyze the transcriptional signatures of cells harvested from the ciliary body and contiguous tissues. Microdissected tissue was dissociated by collagenase digestion and the transcriptomes of individual cells were obtained using a droplet-based scRNA-seq approach. In situ hybridization was used to verify the expression patterns of selected differentially-expressed genes. High quality transcriptomes were obtained from 10,024 cells and unsupervised clustering distinguished 22 cell types. Although efforts were made to specifically isolate the ciliary body, approximately half of the sequenced cells were derived from the adjacent retina. Cluster identities were assigned using expression of canonical markers or cluster-specific genes. The transcriptional signature of cells in the PCE and NPCE were distinct from each other and from cells in contiguous tissues. PCE cell transcriptomes were characterized by genes involved in melanin synthesis and transport proteins such as Slc4a4. Among the most differentially expressed genes in NPCE cells were those encoding members of the Zic family of transcription factors (Zic1, 2, 4), collagen XVIII (Col18a1), and corticotrophin-releasing hormone-binding protein (Crhbp). The ocular melanocyte population was distinguished by expression of the gap junction genes Gjb2 and Gjb6. Two fibroblast signatures were detected in the ciliary body preparation and shown by in situ hybridization to correspond to uveal and scleral populations. This cell atlas for the ciliary body and contiguous layers represents a useful resource that may facilitate studies into the development of the ciliary epithelium, the production of the aqueous and vitreous humors, and the synthesis of the ciliary zonule.

Protective mitochondrial fission induced by stress-responsive protein GJA1-20k.

The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.

Polydnavirus Innexins Disrupt Host Cellular Encapsulation and Larval Maturation.

Polydnaviruses are dsDNA viruses associated with endoparasitoid wasps. Delivery of the virus during parasitization of a caterpillar and subsequent virus gene expression is required for production of an amenable environment for parasitoid offspring development. Consequently, understanding of Polydnavirus gene function provides insight into mechanisms of host susceptibility and parasitoid wasp host range. Polydnavirus genes predominantly are arranged in multimember gene families, one of which is the vinnexins, which are virus homologues of insect gap junction genes, the innexins. Previous studies of Campoletis sonorensis Ichnovirus Vinnexins using various heterologous systems have suggested the four encoded members may provide different functionality in the infected caterpillar host. Here, we expressed two of the members, vnxG and vnxQ2, using recombinant baculoviruses in susceptible host, the caterpillar Heliothis virescens. Following intrahemocoelic injections, we observed that >90% of hemocytes (blood cells) were infected, producing recombinant protein. Larvae infected with a vinnexin-recombinant baculovirus exhibited significantly reduced molting rates relative to larvae infected with a control recombinant baculovirus and mock-infected larvae. Similarly, larvae infected with vinnexin-recombinant baculoviruses were less likely to survive relative to controls and showed reduced ability to encapsulate chromatography beads in an immune assay. In most assays, the VnxG protein was associated with more severe pathology than VnxQ2. Our findings support a role for Vinnexins in CsIV and more broadly Ichnovirus pathology in infected lepidopteran hosts, particularly in disrupting multicellular developmental and immune physiology.

Abstract 2848: Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer

Abstract Background: Hotspot estrogen receptor-α (ER/ERα/ESR1) mutations occur in 30-40% endocrine resistant ER+ breast cancer and are associated with worse outcome. How these mutations facilitate metastasis remains ambiguous. It is necessary to identify a clear mechanism for therapeutic intervention. Methods: ESR1 mutations were detected by ddPCR. Transcriptome data were derived from cell lines and clinical samples. Y537S and D538G genome-edited MCF7 and T47D cell lines were used for in vitro phenotypic characterization. Cell-cell adhesive properties were assessed using calcein-labelled adhesion, spontaneous cell aggregation and ibidi microfluidic assays. Altered cell-cell adhesion genes were validated using qRT-PCR, immunoblot and immunostaining. Desmosome blocking peptides and carbenoxolone were used for blockade of adhesion. ER cistromes were profiled by ChIP-seq. Tail vein injection was performed on athymic nude mice to evaluate metastasis in vivo. Circulating tumor cell (CTC) clustering propensity in vivo was assessed via intracardiac injection followed by CTC microfilter capture. CTC-cluster gene signatures were generated from ER+ breast cancer CTC RNA-seq data. Results: ESR1 mutations were significantly enriched in distant metastases (12/48) vs local (0/27) recurrences, confirming their critical role in promoting metastasis. Transcriptomic analysis revealed altered cell-cell interaction pathways in ESR1 mutant tumors. ESR1 mutant cells formed more compact spheroids in suspension culture, and exhibited stronger cell-cell adhesion in static condition. Under microfluidic condition with physiological shear stress, mutant ESR1 cells derived more and larger clusters, which were prominently preserved from pre-existing clusters. This effect was correlated with increased expression of desmosome and gap junction genes in mutant cells and pharmacological blockade significantly reduced the enhanced cell-cell adhesion. ER ChIP-seq revealed no de novo mutant ER binding sites at the loci of the target genes, suggesting indirect regulation by mutant ER. This was exemplified by a secondary regulation from cFos/AP1 signaling of GJA1 expression, and an epigenetic regulation at DSC1/DSG1 loci through enhanced H3K4me2 and H3K27ac modification. In vivo studies showed ESR1 mutant cells derived more distant metastases, and MCF7 Y537S cells formed larger CTC clusters with increased compactness compared to WT cells. Finally, ER+ CTC-cluster signatures were enriched in ESR1 mutant tumors, suggesting their unique dependence on this pathway during metastasis. Conclusion: Hotspot ESR1 mutations induce expression of multiple desmosome and gap junction genes and confer enhanced cell-cell adhesion, which facilitates breast cancer metastasis via increased CTCs clustering propensity. These findings provide insights to the development of drugs targeting gap junction in ER mutant tumors. Citation Format: Yang Wu, Zheqi Li, Amir Bahreini, Jian Chen, Ye Qin, Kevin M. Levine, Nilgun Tasdemir, Nolan M. Priedigkeit, Li Zhu, George C. Tseng, Yu Jiang, Benjamin Troness, Laki Buluwela, Simak Ali, Spencer Arnesen, Jason Gertz, Ben Ho Park, Jennifer M. Atkinson, Dorraya El-Ashry, Adrian V. Lee, Steffi Oesterreich. Neomorphic cell-cell adhesion reprogramming facilitates metastasis of ESR1 mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2848.

On the potential origin and characteristics of cancer stem cells.

The 'cancer stem cell' hypothesis has pointed to a specific target for new cancer therapies. The hypothesis is based on the observation that only the 'cancer stem cell' among the other heterogeneous cancer cells can sustain the growth of the cancer. The goal is to identify biomarkers of 'cancer stem cells' to distinguish them from the 'cancer non-stem cells' and normal adult tissue-specific stem cells. This analyst posits a hypothesis that, although all cancers originated from a single cell, there exist two types of 'cancer stem cells' either by the 'Stem Cell hypothesis' or from the 'De-differentiation hypothesis'. It is proposed that there exist two different 'cancer stem cells'. Some 'cancer stem cells' (a) lack the expression of connexins or gap junction genes and lack any form of gap junctional intercellular communication (GJIC) or (b) they have the expressed connexin-coded proteins for functional GJIC but are dysfunctional by some expressed oncogene. This is consistent with the Loewenstein hypothesis that a universal characteristic of cancer cells is they do not have growth control, nor terminally differentiate. This review speculates the normal organ-specific adult stem cell, that is 'initiated', is the origin of the 'cancer stem cells' with expressed Oct4A gene and no expressed connexin genes; whereas the other cancer stem cell has no expressed Oct4A genes but expressed connexin gene, whose coded protein is dysfunctional. Hence. both types of 'cancer stem cells' lack GJIC, for two different reasons, the selective therapies have to be different for these different cell types.

Co-exposure to boscalid and TiO2 (E171) or SiO2 (E551) downregulates cell junction gene expression in small intestinal epithelium cellular model and increases pesticide translocation

A recent published study showed that TiO2 (E171) and SiO2 (E551), two widely used nano-enabled food additives, increased the translocation of the commonly used pesticide boscalid by 20% and 30% respectively. Such increased absorption of pesticides due to the presence of engineered nanomaterials (ENMs) in food raises health concerns for these food additives. In this companion study, mRNA expression of genes related to cell junctions in a small intestinal epithelial cellular model after exposure to simulated digestas of fasting food model (phosphate buffer) containing boscalid (150 ppm) with or without either TiO2 or SiO2 (1% w/w) were analyzed. Specific changes in cell barrier function underlying or contributing to the increased translocation of boscalid observed in the previous study were assessed. Results showed that exposure to boscalid alone has no significant effect on cell junction genes, however, co-exposure to boscalid and TiO2 significantly regulated expression of cell-matrix junction focal adhesion-related genes, e.g., downregulating Cav1 (−1.39-fold, p < 0.05), upregulating Cav3 (+ 3.30-fold, p < 0.01) and Itga4 (+ 3.30-fold, p < 0.05). Similarly, co-exposure to boscalid and SiO2 significantly downregulated multiple cell-cell junction genes, including tight junction genes (Cldn1, Cldn11, Cldn16, Cldn18, and Jam3), adherens junction genes (Notch1, Notch3, Pvrl1) and gap junction genes (Gja3 and Gjb2), as well as cell-matrix junction focal adhesion genes (Itga4, Itga6, Itga7). Together, these findings suggest that co-ingestion of boscalid with TiO2 (E171) or SiO2 (E551) could cause weakening of cell junctions and intercellular adhesion, which could result in dysregulation of paracellular transport, and presumably contributed to the previously observed increased translocation of boscalid at the presence of these ENMs. This novel finding raises health safety concerns for such popular food additives.

Abstract GS1-05: Hotspot ESR1 mutations rewire cell-cell adhesome to facilitate breast cancer metastasis

Abstract Background: Estrogen receptor alpha (ER/ESR1) mutations are found in 20-40% of endocrine resistant ER+ metastatic breast cancers, and they are associated with worse outcome. Preclinical studies have shown that they cause ligand-independent growth, resistance to endocrine therapy, and there is growing evidence for a role in metastasis. It is not known how ESR1 mutant cancer cells cause metastases and whether such a mechanism may indicate novel therapeutic options. Methods: ddPCR was used to detect hotspot ESR1 mutations. Transcriptome data were derived from cell line models and clinical samples. For in vitro phenotypic characterization, Y537S and D538G genome-edited MCF7 and T47D cell models from four different labs were used. Cell-cell adhesive properties were assessed using calcein-labelled adhesion, spontaneous cell aggregation and microfluidic aggregation assays. Altered expression of cell-cell adhesion genes detected in RNA seq data was validated via qRT-PCR, immunoblot and immune staining. Functional contributions of desmosome and gap junctions were tested by blocking peptide and carbenoxolone respectively. Mutant ER cistromes were profiled using ChIP-sequencing. Tail vein injection was performed on nude mice to evaluate metastasis in vivo. Mouse lung micro-metastatic foci were quantified using human-specific CK19 staining. Circulating tumor cells (CTCs) clustering propensity in vivo was assessed via intracardiac injection of ESR1 WT and mutant cells into nude mice following CTC microfilter capture. CTCs enumeration from breast cancer patients’ blood samples was performed using CellSearchTM system. Results: We identified a significant enrichment of ESR1 mutations in distant (12/48) vs local (0/27) recurrences, confirming the strong association of mutant ER with metastasis. Transcriptomic analysis revealed altered cell-to-cell interaction pathways in ESR1 mutant tumors compared to ESR1 WT tumors, suggesting a previously undescribed role of ESR1 mutations in reprogramming cell-cell adhesome. ESR1 mutant cells grown in suspension culture revealed more compact multicellular spheroids compared to WT cells. This observation was confirmed under both static and microfluidic conditions, indicative of increased cell-cell interactions. The effect was more pronounced in MCF7 compared to T47D cells, and it correlated with increased expression of multiple desmosome and gap junction pathway genes, which were also significantly enriched in ESR1 mutant tumors. Pharmacological blockade of desmosome and gap junctions significantly rescued enhanced cell-cell adhesion in ESR1 mutant cells. Mechanistically, our ER ChIP-seq did not identify any gained mutant ER binding sites in proximity to cell-cell adhesion gene loci, indicating indirect regulation by mutant ER. Consistent with this, expression of Connexin 43, one of the top upregulated gap junction components, was induced by cFOS found to be highly upregulated in ESR1 mutant cells. Tail vein injection of ESR1-mutant cells derived more distant macro- (MCF7) and micro- (T47D) metastases. Given increasing evidence for role of cell-cell attachment in CTC phenotypes, we tested CTC formation for ESR1 WT and mutant cells. In vivo studies showed MCF7 Y537S ESR1 mutant cells formed larger multi-cellular CTC clusters with increased compactness compared to WT CTCs. These preclinical data translated to clinical observation, where we observed an enrichment of CTC clusters in patients with ESR1 mutant-metastatic breast cancers. Conclusion: Hotspot ESR1 mutations induce expression of multiple desmosome and gap junction genes and confer increased cell-cell adhesion, which facilitate breast cancer metastasis via increased CTCs clustering propensity. These findings might guide approaches to test potential repurpose of drugs targeting gap junction in ER mutant tumors. Citation Format: Zheqi Li, Yang Wu, Amir Bahreini, Jian Chen, Ye Qin, Kevin M. Levine, Nilgun Tasdemir, Nolan Priedigkeit, Li Zhu, George C. Tseng, Yu Jiang, Benjamin Troness, Laki Buluwela, Simak Ali, Spencer Arnesen, Jason Gertz, Ben H. Park, Qiang Zhang, Lorenzo Gerratana, Andrew Davis, Jennifer M. Atkinson, Dorraya El-Ashry, Massimo Cristofanilli, Adrian V. Lee, Steffi Oesterreich. Hotspot ESR1 mutations rewire cell-cell adhesome to facilitate breast cancer metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS1-05.

Atrial fibrillation\u2014a complex polygenetic disease

Atrial fibrillation (AF) is the most common type of arrhythmia. Epidemiological studies have documented a substantial genetic component. More than 160 genes have been associated with AF during the last decades. Some of these were discovered by classical linkage studies while the majority relies on functional studies or genome-wide association studies. In this review, we will evaluate the genetic basis of AF and the role of both common and rare genetic variants in AF. Rare variants in multiple ion-channel genes as well as gap junction and transcription factor genes have been associated with AF. More recently, a growing body of evidence has implicated structural genes with AF. An increased burden of atrial fibrosis in AF patients compared with non-AF patients has also been reported. These findings challenge our traditional understanding of AF being an electrical disease. We will focus on several quantitative landmark papers, which are transforming our understanding of AF by implicating atrial cardiomyopathies in the pathogenesis. This new AF research field may enable better diagnostics and treatment in the future.