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.

Molecular and morphological approach to study the innexin gap junctions in Rhynchosciara americana

Gap junctions mediate communication between adjacent cells and are fundamental to the development and homeostasis in multicellular organisms. In invertebrates, gap junctions are formed by transmembrane proteins called innexins. Gap junctions allow the passage of small molecules through an intercellular channel, between a cell and another adjacent cell. The dipteran Rhynchosciara americana has contributed to studying the biology of invertebrates and the study of the interaction and regulation of genes during biological development. Therefore, this paper aimed to study the R. americana innexin-2 by molecular characterization, analysis of the expression profile and cellular localization. The molecular characterization results confirm that the message is from a gap junction protein and analysis of the expression and cellular localization profile shows that innexin-2 can participate in many physiological processes during the development of R. americana .

Connexins evolved after early chordates lost innexin diversity

Gap junction channels are formed by two unrelated protein families. Non-chordates use the primordial innexins, while chordates use connexins that superseded the gap junction function of innexins. Chordates retained innexin-homologs, but N-glycosylation prevents them from forming gap junctions. It is puzzling why chordates seem to exclusively use the new gap junction protein and why no chordates should exist that use non-glycosylated innexins to form gap junctions. Here, we identified glycosylation sites of 2270 innexins from 152 non-chordate and 274 chordate species. Among all chordates, we found not a single innexin without glycosylation sites. Surprisingly, the glycosylation motif is also widespread among non-chordate innexins indicating that glycosylated innexins are not a novelty of chordates. In addition, we discovered a loss of innexin diversity during the early chordate evolution. Most importantly, the most basal living chordates, which lack connexins, exclusively possess innexins with glycosylation sites. A bottleneck effect might thus explain why connexins have become the only protein used to form chordate gap junctions.

Changes in Stress-Mediated Markers in a Human Cardiomyocyte Cell Line under Hyperglycemia

Diabetes is a major risk factor for cardiovascular diseases, especially cardiomyopathy, a condition in which the smooth muscles of the heart become thick and rigid, affecting the functioning of cardiomyocytes, the contractile cells of the heart. Uncontrolled elevated glucose levels over time can result in oxidative stress, which could lead to inflammation and altered epigenetic mechanisms. In the current study, we investigated whether hyperglycemia can modify cardiac function by directly affecting these changes in cardiomyocytes. To evaluate the adverse effect of high glucose, we measured the levels of gap junction protein, connexin 43, which is responsible for modulating cardiac electric activities and Troponin I, a part of the troponin complex in the heart muscles, commonly used as cardiac markers of ischemic heart disease. AC16 human cardiomyocyte cells were used in this study. Under hyperglycemic conditions, these cells demonstrated altered levels of connexin 43 and Troponin-I after 24 h of exposure. We also examined hyperglycemia induced changes in epigenetic markers: H3K9me1, Sirtuin-1 (SIRT1), and histone deacetylase (HDAC)-2 as well as in inflammatory and stress-related mediators, such as heat shock protein (HSP)-60, receptor for advanced glycation end products (RAGE), toll-like receptor (TLR)-4, high mobility group box (HMGB)-1 and CXC chemokine receptor (CXCR)-4. Cardiomyocytes exposed to 25mM glucose resulted in the downregulation of HSP60 and SIRT1 after 48 h. We further examined that hyperglycemia mediated the decrease in the gap junction protein CX43, as well as CXC chemokine receptor CXCR4 which may affect the physiological functions of the cardiomyocytes when exposed to high glucose for 24 and 48 h. Upregulated expression of DNA-binding nuclear protein HMGB1, along with changes in histone methylation marker H3K9me1 have demonstrated hyperglycemia-induced damage to cardiomyocyte at 24 h of exposure. Our study established that 24 to 48 h of hyperglycemic exposure could stimulate stress-mediated inflammatory mediators in cardiomyocytes in vitro. These stress-related changes in hyperglycemia-induced cardiomyocytes may further initiate an increase in injury markers which eventually could alter the epigenetic processes. Therefore, epigenetic and inflammatory mechanisms in conjunction with alterations in a downstream signaling pathway could have a direct effect on the functionality of the cardiomyocytes exposed to high glucose during short and long-term exposures.

Cis-acting mutation affecting GJA5 transcription is underlying the Melanotic within-feather pigmentation pattern in chickens

Melanotic (Ml) is a mutation in chickens that extends black (eumelanin) pigmentation in normally brown or red (pheomelanin) areas, thus affecting multiple within-feather patterns [J. W. Moore, J. R. Smyth Jr, J. Hered. 62, 215–219 (1971)]. In the present study, linkage mapping using a back-cross between Dark Cornish (Ml/Ml) and Partridge Plymouth Rock (ml+/ml+) chickens assigned Ml to an 820-kb region on chromosome 1. Identity-by-descent mapping, via whole-genome sequencing and diagnostic tests using a diverse set of chickens, refined the localization to the genomic region harboring GJA5 encoding gap-junction protein 5 (alias connexin 40) previously associated with pigmentation patterns in zebrafish. An insertion/deletion polymorphism located in the vicinity of the GJA5 promoter region was identified as the candidate causal mutation. Four different GJA5 transcripts were found to be expressed in feather follicles and at least two showed differential expression between genotypes. The results showed that Melanotic constitutes a cis-acting regulatory mutation affecting GJA5 expression. A recent study established the melanocortin-1 receptor (MC1R) locus and the interaction between the MC1R receptor and its antagonist agouti-signaling protein as the primary mechanism underlying variation in within-feather pigmentation patterns in chickens. The present study advances understanding the mechanisms underlying variation in plumage color in birds because it demonstrates that the activity of connexin 40/GJA5 can modulate the periodic pigmentation patterns within individual feathers.

Analysis of Rod/Cone Gap Junctions from the Reconstruction of Mouse Photoreceptor Terminals

Using serial blockface-scanning electron microscopy (SBF-SEM) and focused ion beam-scanning electron microscopy (FIB-SEM), combined with confocal microscopy for the gap junction protein Cx36, we reconstructed mouse photoreceptor terminals and located the gap junctions between them. An exuberant spray of fine telodendria extends from each cone pedicle (including blue cones) to contact 40-50 nearby rod spherules where Cx36 clusters were located, close to the mouth of the synaptic opening. There were approximately Cx36 clusters per cone 50 pedicle and 2-3 per rod spherule. We were unable to detect rod/rod or cone/cone coupling. Thus, rod/cone coupling accounts for nearly all gap junctions between photoreceptors. Our calculations suggest a mean of 82 Cx36 channels between a rod/cone pair of which 25% are open at rest. Rod/cone gap junctions are modulated by dopamine. Comparing our morphological calculations of maximum coupling to previous physiological results suggests that dopamine antagonists can drive rod/cone gap junctions to a surprisingly high open probability, approaching 100%.

Xanthine oxidase inhibitor febuxostat reduces atrial fibrillation susceptibility by inhibition of oxidized CaMKII in Dahl salt-sensitive rats

Oxidative stress could be a possible mechanism and a therapeutic target of atrial fibrillation (AF). However, the effects of the xanthine oxidase (XO) inhibition for AF remain to be fully elucidated. We investigated the effects of a novel XO inhibitor febuxostat on AF compared to allopurinol in hypertension rat model. Five-week-old Dahl salt-sensitive rats were fed to either low-salt (LS) (0.3% NaCl) or high-salt (HS) (8% NaCl) diet. After 4 weeks of diet, HS diet rats were divided into 3 groups: orally administered to vehicle (HS-C), febuxostat (5mg/kg/day) (HS-F), or allopurinol (50mg/kg/day) (HS-A). After 4 weeks of treatment, systolic blood pressure was significantly higher in HS-C than LS, and it was slightly but significantly decreased by treatment with each XO inhibitor. AF duration was significantly prolonged in HS-C compared with LS, and significantly suppressed in both HS-F and HS-A (LS; 5.8±3.5 sec, HS-C; 33.9±23.7 sec, HS-F; 15.0±14.1 sec, HS-A; 20.1±11.9 sec: P<0.05). Ca2+ spark frequency was obviously increased in HS-C rats and reduced in the XO inhibitor-treated rats, especially in HS-F group. Western blotting revealed that the atrial expression levels of methionine 281/282-oxidized Ca2+/Calmodulin-dependent kinase II and serine 2814-phosphorylated ryanodine receptor 2 were significantly increased in HS-C, and those were suppressed in HS-F and HS-A. Decreased expression of gap junction protein connexin 40 in HS-C was partially restored by treatment with each XO inhibitor. In conclusion, XO inhibitor febuxostat, as well as allopurinol, could reduce hypertension-related increase in AF perpetuation by restoring Ca2+ handling and gap junction.

Differential expression of gap junction protein beta 2 in human epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer (1). We performed discovery of genes associated with epithelial ovarian cancer and of the high-grade serous ovarian cancer (HGSC) subtype, using published microarray data (2, 3) to compare global gene expression profiles of normal ovary or fallopian tube with that of primary tumors from women diagnosed with epithelial ovarian cancer or HGSC. We identified the gene encoding gap junction protein beta 2, GJB2, as among the genes whose expression was most different in epithelial ovarian cancer as compared to the normal fallopian tube. GJB2 expression was significantly higher in high-grade serous ovarian tumors relative to normal fallopian tube. GJB2 expression correlated with overall survival in patients with ovarian cancer. These data indicate that expression of GJB2 is perturbed in epithelial ovarian cancers broadly and in ovarian cancers of the HGSC subtype. GJB2 may be relevant to pathways underlying ovarian cancer initiation (transformation) or progression.

Omega-3 PUFAs Suppress IL-1β-Induced Hyperactivity of Immunoproteasomes in Astrocytes

The role of immunoproteasome (iP) in astroglia, the cellular component of innate immunity, has not been clarified. The results so far indicate that neuroinflammation, a prominent hallmark of Alzheimer’s disease, strongly activates the iP subunits expression. Since omega-3 PUFAs possess anti-inflammatory and pro-resolving activity in the brain, we investigated the effect of DHA and EPA on the gene expression of constitutive (β1 and β5) and inducible (iβ1/LMP2 and iβ5/LMP7) proteasome subunits and proteasomal activity in IL-1β-stimulated astrocytes. We found that both PUFAs downregulated the expression of IL-1β-induced the iP subunits, but not the constitutive proteasome subunits. The chymotrypsin-like activity was inhibited in a dose-dependent manner by DHA, and much strongly in the lower concentration by EPA. Furthermore, we established that C/EBPα and C/EBPβ transcription factors, being the cis-regulatory element of the transcription complex, frequently activated by inflammatory mediators, participate in a reduction in the iP subunits’ expression. Moreover, the expression of connexin 43 the major gap junction protein in astrocytes, negatively regulated by IL-1β was markedly increased in PUFA-treated cells. These findings indicate that omega-3 PUFAs attenuate inflammation-induced hyperactivity of iPs in astrocytes and have a beneficial effect on preservation of interastrocytic communication by gap junctions.