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.

First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization

In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze–fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C–terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

The Concept of “Cancer Stem Cells” in the Context of Classic Carcinogenesis Hypotheses and Experimental Findings

In this Commentary, the operational definition of cancer stem cells or cancer initiating cells includes the ability of certain cells, found in a heterogeneous mixture of cells within a tumor, which are able to sustain growth of that tumor. However, that concept of cancer stem cells does not resolve the age-old controversy of two opposing hypotheses of the origin of the cancer, namely the stem cell hypothesis versus the de-differentiation or re-programming hypothesis. Moreover, this cancer stem concept has to take into account classic experimental observations, techniques, and concepts, such as the multi-stage, multi-mechanism process of carcinogenesis; roles of mutagenic, cytotoxic and epigenetic mechanisms; the important differences between errors of DNA repair and errors of DNA replication in forming mutations; biomarkers of known characteristics of normal adult organ-specific stem cells and of cancer stem cells; and the characteristics of epigenetic mechanisms involved in the carcinogenic process. In addition, vague and misleading terms, such as carcinogens, immortal and normal cells have to be clarified in the context of current scientific facts. The ultimate integration of all of these historic factors to provide a current understanding of the origin and characteristics of a cancer stem cell, which is required for a rational strategy for prevention and therapy for cancer, does not follow a linear path. Lastly, it will be speculated that there exists evidence of two distinct types of cancer stem cells, one that has its origin in an organ-specific adult stem cell that is ‘initiated’ in the stem cell stage, expressing the Oct4A gene and not expressing any connexin gene or having functional gap junctional intercellular communication (GJIC). The other cancer stem cell is derived from a stem cell that is initiated early after the Oct4A gene is suppressed and the connexin gene is expressed, which starts early differentiation, but it is blocked from terminal differentiation.

PDGF-AA promotes cell-to-cell communication in osteocytes through PI3K/Akt signaling pathway

Osteocytes are the main sensitive cells in bone remodeling due to their potent functional cell processes from the mineralized bone matrix to the bone surface and the bone marrow. Neighboring osteocytes communicate with each other by these cell processes to achieve molecular exchange through gap junction channels. Platelet-derived growth factor-AA (PDGF-AA) has been reported to enhance bone tissue remodeling by promoting cell proliferation, migration, and autocrine secretion in osteoid cell linage. However, the effect of PDGF-AA on intercellular communication between osteocytes is still unclear. In the present study, we elucidated that PDGF-AA could enhance the formation of dendritic processes of osteocytes and the gap junctional intercellular communication by promoting the expression of connexin43 (Cx43). This modulation process was mainly dependent on the activation of phosphorylation of Akt protein by phosphatidylinositol 3-kinase (PI3K)/Akt (also known as protein kinase B, PKB) signaling. Inhibition of PI3K/Akt signaling decreased the Cx43 expression induced by PDGF-AA. These results establish a bridge between PDGF-AA and cell–cell communication in osteocytes, which could help us understand the molecular exchange between bone cells and fracture healing.

Ouabain Enhances Gap Junctional Intercellular Communication by Inducing Paracrine Secretion of Prostaglandin E2

Ouabain is a cardiac glycoside that has been described as a hormone, with interesting effects on epithelial physiology. We have shown previously that ouabain induces gap junctional intercellular communication (GJIC) in wild, sensitive cells (MDCK-S), but not in cells that have become insensitive (MDCK-I) by modifying their Na+-K+-ATPase. We have also demonstrated that prostaglandin E2 (PGE2) is able to induce increased GJIC by a mechanism other than ouabain, that does not depend on Na+-K+-ATPase. In this work we show, by dye transfer assays, that when MDCK-S and MDCK-I are randomly mixed, to form monolayers, the latter stablish GJIC, because of stimulation by a compound released to the extracellular media, by MDCK-S cells, after treatment with ouabain, as evidenced by the fact that monolayers of only MDCK-I cells, treated with a conditioned medium (CM) that is obtained after incubation of MDCK-S monolayers with ouabain, significantly increase their GJIC. The further finding that either (1) pre-treatment with COX-2 inhibitors or (2) addition to CM of antagonists of EP2 receptor abolish CM’s ability to induce GJIC in MDCK-I monolayers indicate that PGE2 is the GJIC-inducing compound. Therefore, these results indicate that, in addition to direct stimulation, mediated by Na+-K+-ATPase, ouabain enhances GJIC indirectly through the paracrine production of PGE2.

Specific N-cadherin–dependent pathways drive human breast cancer dormancy in bone marrow

The challenge for treating breast cancer (BC) is partly due to long-term dormancy driven by cancer stem cells (CSCs) capable of evading immune response and resist chemotherapy. BC cells show preference for the BM, resulting in poor prognosis. CSCs use connexin 43 (Cx43) to form gap junctional intercellular communication with BM niche cells, fibroblasts, and mesenchymal stem cells (MSCs). However, Cx43 is an unlikely target to reverse BC dormancy because of its role as a hematopoietic regulator. We found N-cadherin (CDH2) and its associated pathways as potential drug targets. CDH2, highly expressed in CSCs, interacts intracellularly with Cx43, colocalizes with Cx43 in BC cells within BM biopsies of patients, and is required for Cx43-mediated gap junctional intercellular communication with BM niche cells. Notably, CDH2 and anti-apoptotic pathways maintained BC dormancy. We thereby propose these pathways as potential pharmacological targets to prevent dormancy and chemosensitize resistant CSCs.

Seeing Is Believing: Gap Junctions in Motion

Gap junctional intercellular communication (GJIC) channels between cells are composed of connexin proteins that form hexamers (connexons) in adjacent plasma membranes [...]

Prostaglandin E2 Enhances Gap Junctional Intercellular Communication in Clonal Epithelial Cells

Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.

Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

AIM: To study the effect of zymosan, a ligand found on the surface of fungi, on gap junctional intercellular communication (GJIC) in cultured human corneal fibroblasts (HCFs). METHODS: Zymosan was added to the medium of cultured HCFs with or without the administration of mitogen-activated protein kinase (MAPK) inhibitors or the inhibitor kappa B kinase 2 (IKK2) inhibitor IV. The protein and mRNA levels of connexin 43 (Cx43) in HCFs were measured by Western blot, immunofluorescence, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The GJIC activity was tested using a dye-coupling assay. RESULTS: The reduction of Cx43 protein and mRNA levels as well as a significant decrease in GJIC activity were observed in cultured HCFs when zymosan was added into the culture medium. Compared with controls (no zymosan), the protein level of Cx43 was reduced by 45% and 54% in the presence of zymosan at 200 and 600 μg/mL, respectively (P<0.05); and it was reduced by 45%, 48%, and 75% in the presence of zymosan (600 μg/mL) for 24, 36, and 48h, respectively (P<0.05). The mRNA expression of Cx43 was reduced by 98% in the presence of zymosan (P<0.05). The effects of zymosan on Cx43 expression and GJIC activity were attenuated by the administration of PD98059 [an extracellular signal-regulated kinase (ERK) signaling inhibitor] (P<0.05), c-Jun NH2-terminal kinase (JNK) inhibitor II (P<0.05), and IKK2 inhibitor IV (P<0.05). CONCLUSION: Zymosan inhibits the activity of GJIC in cultured HCFs. This effect is likely regulated via the nuclear factor-κB (NF-κB), MAPK/ERK, and JNK signaling pathways. The inhibitory effects of zymosan on Cx43 expression and GJIC activity in HCFs may induce damage of corneal stroma during corneal fungal infection.