Connexin 43 confers chemoresistance through activating PI3K

Circumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1–a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide–inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit β (PIK3CB, also called PI3Kβ or p110β), suggesting that Cx43 activates PIK3CB/p110β independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110β, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110β-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110β together is an effective therapeutic approach for overcoming chemoresistance.

LRP6 modulates the phosphorylation of Cx43 via Gαs in ventricular tachycardia of myocardial infarction

Background Ventricular tachycardia (VT) and ventricular fibrillation are the most causes of early death in patients with myocardial infarction (MI). This study was aimed to explore whether LRP6 and its upstream genes circRNA1615 and miR-152-3p modulated the phosphorylation of Connexin-43 (Cx43) via Gαs in ventricular tachycardia of MI. Method we constructed the hypoxia cardiomyocyte model and AMI mice, and explored the modulation relationship of LRP6 and its upstream genes circRNA1615 and miR-152-3p. In addition, the immunoblot analysis with monoclonal and polyclonal antibodies were used to detect whether LRP6 and Cx43 were phosphorylated, further investigated that the LRP6 regulated the phosphorylation of its downstream target Cx43 via G-protein alpha subunit Gαs by using cell transfection, FISH assay, HE staining, RT-qPCR, and Western blot techniques. Result LRP6 mRNA expression was significantly reduced in AMI group compared with the control group. Hypoxia could inhibit the protein and phosphorylation levels of LRP6 and Cx43. The expression of circRNA1615 in AMI mice was significantly decreased, but overexpression of circRNA1615 significantly reversed it. Also overexpression of circRNA1615 could weaken the effect of miR-152-3p mimic, and the miR-152-3p mimic increased the hypoxia injury of LRP6 and Cx43, further LRP6 interference fragments could aggravate hypoxia injury of Cx43. The overexpression of LRP6 could significantly increase the protein level and phosphorylation level of Cx43, but the interference with LRP6 showed the opposite trend. Noticeably, the interference with Gαs weakened the protein and phosphorylation levels of Cx43, however, the interference with LRP6 further inhibited the protein and phosphorylation levels of Cx43. Finally, the transcriptions of circRNA1615 and LRP6 were inhibited in AMI, but the transcription of miR-152-3p was promoted, and the overexpression of circRNA1615 could weaken the damage effect and VT of AMI. Conclusion LRP6 and its upstream genes circRNA1615 and miR-152-3p modulated the phosphorylation of Cx43 via Gαs in ventricular tachycardia of myocardial infarction.

Connexin 43: A Target for the Treatment of Inflammation in Secondary Complications of the Kidney and Eye in Diabetes

Of increasing prevalence, diabetes is characterised by elevated blood glucose and chronic inflammation that precedes the onset of multiple secondary complications, including those of the kidney and the eye. As the leading cause of end stage renal disease and blindness in the working population, more than ever is there a demand to develop clinical interventions which can both delay and prevent disease progression. Connexins are membrane bound proteins that can form pores (hemichannels) in the cell membrane. Gated by cellular stress and injury, they open under pathophysiological conditions and in doing so release ‘danger signals’ including adenosine triphosphate into the extracellular environment. Linked to sterile inflammation via activation of the nod-like receptor protein 3 inflammasome, targeting aberrant hemichannel activity and the release of these danger signals has met with favourable outcomes in multiple models of disease, including secondary complications of diabetes. In this review, we provide a comprehensive update on those studies which document a role for aberrant connexin hemichannel activity in the pathogenesis of both diabetic eye and kidney disease, ahead of evaluating the efficacy of blocking connexin-43 specific hemichannels in these target tissues on tissue health and function.

GJA1-20k, an internally translated isoform of Connexin 43, is an actin capping protein

Previously, we identified that GJA1-20k, an internally translated isoform of Connexin 43, mediates an actin-dependent protective form of mitochondrial fission (Shimura, Nuebel et al. 2021). We found that when GJA1-20k is present, bands of actin surround mitochondria at locations enriched with GJA1-20k, inducing mitochondrial fission which generates less oxygen free radicals, protecting hearts subjected to ischemia-reperfusion injury. Here, we report that GJA1-20k is a direct actin binding protein and thereby identify the mechanism by which GJA1-20k is able to recruit and stabilize actin filaments around the mitochondria. Surprisingly, GJA1-20k functions as a canonical actin capping protein, producing both truncated actin puncta and stabilized actin filaments. GJA1-20k contains an RPEL-like actin binding motif, and we confirm with both computational modeling and biochemistry, that this domain is crucial for actin capping. The actin capping functionality of GJA1-20k adds GJA1-20k to the family of proteins that regulate actin dynamics. As a stress responsive protein, GJA1-20k can help explain cytoskeletal dependent responses to cellular stress, from delivery of channels to affecting mitochondrial size and function.

Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

Connexin 43 Gene Ablation Does Not Alter Human Pluripotent Stem Cell Germ Lineage Specification

During embryonic germ layer development, cells communicate with each other and their environment to ensure proper lineage specification and tissue development. Connexin (Cx) proteins facilitate direct cell–cell communication through gap junction channels. While previous reports suggest that gap junctional intercellular communication may contribute to germ layer formation, there have been limited comprehensive expression analyses or genetic ablation studies on Cxs during human pluripotent stem cell (PSC) germ lineage specification. We screened the mRNA profile and protein expression patterns of select human Cx isoforms in undifferentiated human induced pluripotent stem cells (iPSCs), and after directed differentiation into the three embryonic germ lineages: ectoderm, definitive endoderm, and mesoderm. Transcript analyses by qPCR revealed upregulation of Cx45 and Cx62 in iPSC-derived ectoderm; Cx45 in mesoderm; and Cx30.3, Cx31, Cx32, Cx36, Cx37, and Cx40 in endoderm relative to control human iPSCs. Generated Cx43 (GJA1) CRISPR-Cas9 knockout iPSCs successfully differentiated into cells of all three germ layers, suggesting that Cx43 is dispensable during directed iPSC lineage specification. Furthermore, qPCR screening of select Cx transcripts in our GJA1-/- iPSCs showed no significant Cx upregulation in response to the loss of Cx43 protein. Future studies will reveal possible compensation by additional Cxs, suggesting targets for future CRISPR-Cas9 ablation studies in human iPSC lineage specification.

Osteocytic Connexin Hemichannels Modulate Oxidative Bone Microenvironment and Breast Cancer Growth

Osteocytes, the most abundant bone cell types embedded in the mineral matrix, express connexin 43 (Cx43) hemichannels that play important roles in bone remodeling and osteocyte survival. Estrogen deficiency decreases osteocytic Cx43 hemichannel activity and causes a loss in osteocytes’ resistance to oxidative stress (OS). In this study, we showed that OS reduced the growth of both human (MDA-MB-231) and murine (Py8119) breast cancer cells. However, co-culturing these cells with osteocytes reduced the inhibitory effect of OS on breast cancer cells, and this effect was ablated by the inhibition of Cx43 hemichannels. Py8119 cells were intratibially implanted in the bone marrow of ovariectomized (OVX) mice to determine the role of osteocytic Cx43 hemichannels in breast cancer bone metastasis in response to OS. Two transgenic mice overexpressing dominant-negative Cx43 mutants, R76W and Δ130-136, were adopted for this study; the former inhibits gap junctions while the latter inhibits gap junctions and hemichannels. Under normal conditions, Δ130-136 mice had significantly more tumor growth in bone than that in WT and R76W mice. OVX increased tumor growth in R76W but had no significant effect on WT mice. In contrast, OVX reduced tumor growth in Δ130-136 mice. To confirm the role of OS, WT and Δ130-136 mice were administered the antioxidant N-acetyl cysteine (NAC). NAC increased tumor burden and growth in Δ130-136 mice but not in WT mice. Together, the data suggest that osteocytes and Cx43 hemichannels play pivotal roles in modulating the oxidative microenvironment and breast cancer growth in the bone.

Acute Methylglyoxal-Induced Damage In Blood-Brain Barrier And Hippocampal Tissue

Methylglyoxal (MG) is a reactive dicarbonyl compound formed mostly by the glycolytic pathway. Elevated blood glucose levels can cause MG accumulation in plasma and cerebrospinal fluid diabetes mellitus and Alzheimer’s disease, where the high reactivity of MG leads to modification of proteins and other biomolecules, generating advanced glycation end products (AGEs) appointed as mediators in those neurodegenerative diseases. Herein, we investigated the blood-brain barrier (BBB) integrity and astrocyte response in the hippocampus to acute insult induced by MG, administered ICV in rats. Seventy-two hours later, a loss of BBB integrity was observed, as assessed by the entry of Evans dye into brain tissue and albumin in the CSF, as well as a decrease of aquaporin-4 and connexin-43 in hippocampal tissue. MG did not induce changes in hippocampal contents of RAGE in this short interval, but decreased the expression of S100B, an astrocyte secreted protein that binds RAGE. The expressions of two important transcription factors of antioxidant response - NfkB and Nrf2, were not changed. However, hemeoxigenase-1 was upregulated in MG-treated group. This data corroborates with the idea that astrocytes, the main cells responsible for MG clearance, are targets of MG toxicity and that BBB dysfunction induced by this compound may contribute to behavioral and cognitive alterations observed in these animals.