Mitochondria, the dynamic organelles responsible for energy production and cellular metabolism, have the metabolic function of extracting energy from nutrients and synthesizing crucial metabolites. Nevertheless, recent research unveils that intercellular mitochondrial transfer by tunneling nanotubes, tumor microtubes, gap junction intercellular communication, extracellular vesicles, endocytosis and cell fusion may regulate mitochondrial function within recipient cells, potentially contributing to disease treatment, such as nonalcoholic steatohepatitis, glioblastoma, ischemic stroke, bladder cancer and neurodegenerative diseases. This review introduces the principal approaches to intercellular mitochondrial transfer and examines its role in various diseases. Furthermore, we provide a comprehensive overview of the inhibitors and activators of intercellular mitochondrial transfer, offering a unique perspective to illustrate the relationship between intercellular mitochondrial transfer and diseases.

The in vitro maturation (IVM) quality of oocytes is directly related to the subsequent developmental potential of embryos and a fundamental of in vitro embryo production. However, conventional IVM methods fail to maintain the gap-junction intercellular communication (GJIC) between cumulus-oocyte complexes (COCs), which leads to insufficient oocyte maturation. Herein, we investigated the effects of three different three-dimensional (3D) culture methods on oocyte development in vitro, optimized of the alginate-hydrogel embedding method, and assessed the effects of the alginate-hydrogel embedding method on subsequent embryonic developmental potential of oocytes after IVM and parthenogenetic activation (PA). The results showed that Matrigel embedding and alginate-hydrogel embedding benefited the embryonic developmental potential of oocytes after IVM and PA. With the further optimization of alginate-hydrogel embedding, including crosslinking and decrosslinking of parameters, we established a 3D culture system that can significantly increase oocyte maturation and the blastocyst rate of embryos after PA (27.2 ± 1.5 vs 36.7 ± 2.8, P < 0.05). This 3D culture system produced oocytes with markedly increased mitochondrial intensity and membrane potential, which reduced the abnormalities of spindle formation and cortical granule distribution. The alginate-hydrogel embedding system can also remarkably enhance the GJIC between COCs. In summary, based on alginate-hydrogel embedding, we established a 3D culture system that can improve the IVM quality of porcine oocytes, possibly by enhancing GJIC.

OBJECTIVE: To explore the influence of PDGF-AA on cell communication between human dental pulp stem cells (DPSCs) by characterizing gap junction intercellular communication (GJIC) and its potential biomechanical mechanism.
METHODS: Quantitative real-time PCR was used to measure connexin family member expression in DPSCs. Cell migration and CCK-8 assays were utilized to examine the influence of PDGF-AA on DPSC migration and proliferation. A scrape loading/dye transfer assay was applied to evaluate GJIC triggered by PDGF-AA, a PI3K/Akt signalling pathway blocker (LY294002) and a PDGFR-α blocker (AG1296). Western blotting and immunofluorescence were used to test the expression and distribution of the Cx43 and p-Akt proteins in DPSCs. Scanning electron microscopy (SEM) and immunofluorescence were used to observe the morphology of GJIC in DPSCs.
RESULTS: PDGF-AA promoted gap junction formation and intercellular communication between human dental pulp stem cells. PDGF-AA upregulates the expression of Cx43 to enhance gap junction formation and intercellular communication. PDGF-AA binds to PDGFR-α and activates PI3K/Akt signalling to regulate cell communication.
CONCLUSIONS: This research demonstrated that PDGF-AA can enhance Cx43-mediated GJIC in DPSCs via the PDGFR-α/PI3K/Akt axis, which provides new cues for dental pulp regeneration from the perspective of intercellular communication.

Gap junction intercellular communication (GJIC) among granulosa cells plays an important role in folliculogenesis, and it is temporal-spatially regulated during follicular development. Connexin (Cx) proteins predominantly form the basal structure of gap junctions in granulosa cells. In our study, immunohistochemical analysis revealed that Cx43 is the most widely expressed connexin in porcine follicles, especially among the large antral follicles. With application of insulin on porcine granulosa cells, we found that insulin significantly facilitated the protein level of Cx43, not mRNA level. This process is dependent on the phosphorylated activities of AKT and Erk since selective AKT and Erk inhibitors, LY294002 and U0126, respectively, hampered the potential of insulin to up-regulate Cx43 protein expression. As a consequence, the insulin-enhanced Cx43-couple GJIC activity in porcine granulosa cells was corresponding attenuated by the administration of LY294002 and U0126. Our findings provide a new insight into the molecular mechanisms by which insulin mediates cell-cell communication in porcine granulosa cells and sheds light on nutrition-reproduction interactions.

Gap junction intercellular communication (GJIC) is essential for regulating the development of the organism and sustaining the internal environmental homeostasis of multi-cellular tissue. Fibroblast growth factor 8 (FGF8), an indispensable regulator of the skeletal system, is implicated in regulating chondrocyte growth, differentiation, and disease occurrence. However, the influence of FGF8 on GJIC in chondrocytes is not yet known. The study aims to investigate the role of FGF8 on cell-cell communication in chondrocytes and its underlying biomechanism. We found that FGF8 facilitated cell-cell communication in living chondrocytes by the up-regulation of connexin43 (Cx43), the major fundamental component unit of gap junction channels in chondrocytes. FGF8 activated p38-MAPK signaling to increase the expression of Cx43 and promote the cell-cell communication. Inhibition of p38-MAPK signaling impaired the increase of Cx43 expression and cell-cell communication induced by FGF8, indicating the importance of p38-MAPK signaling. These results help to understand the role of FGF8 on cell communication and provide a potential cue for the treatment of cartilage diseases.

BACKGROUND: The gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined.
METHODS: Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance.
RESULTS: The results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells.
CONCLUSIONS: BTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.

As a common environmental pollutant, cadmium (Cd) causes damage to many organs of the body. Gap junction intercellular communication (GJIC) represents one of the most important routes of rapid signaling between cells. However, the mechanisms underlying GJIC\'s role in hepatotoxicity induced by Cd remain unknown. We established a Cd poisoning model in vitro by co-culturing Cd-exposed and unexposed hepatocytes and found that 18β-glycyrrhetinic acid (GA), a GJIC inhibitor, can effectively reduce the apoptosis rate of healthy cells co-cultured with apoptotic cells treated with Cd. We also found that anti-FasL antibody had the same effect. However, in mono-cultured cells, GA treatment in combination with Cd was found to aggravate the damage induced by Cd exposure, increase the level of oxidative stress and protein expression of HO-1, decrease the mitochondrial membrane potential, incur more serious morphological damage to mitochondria than Cd treatment alone. Moreover, compared with Cd-only exposure, GA and Cd co-treatment further increased the expression levels of the apoptosis-related proteins Fas, FasL, FADD and the ratio of Bax/Bcl-2, inhibited the protein expression of ASK1 and Daxx. We also found that the protein expression of Daxx in siFADD + Cd hepatocytes was significantly higher than in Cd-treated cells. Thus, our study suggests that gap junction inhibition may play a dual role in Cd-induced cell damage by inhibiting the transmission of death signals from damaged cells to healthy cells but also aggravating the transmission of death signals between damaged cells, and that the Fas/FasL-mediated death receptor pathway may play an important role in this process.

Aim: To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis. Materials & methods: Previously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid were used as cell culture substrates to control cell adhesion at the nanoscale. In vitro chondrogenesis of mesenchymal stem cells was conducted on the nanopatterns. Cohesion and GJIC were evaluated in cell condensates. Results: Mechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling. Conclusion: Cell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.

Dioscin (Dio), steroid saponin, exists in several medicinal herbs with potent anticancer efficacy. This study aimed to explore the effect of Dio on the immune-related modulation and synergistic therapeutic effects of the herpes simplex virus thymidine kinase/ganciclovir (HSV-Tk/GCV) suicide gene therapy system in murine melanoma, thereby providing a research basis to improve the potential immunomodulatory mechanism underlying combination therapy. Using both in vitro and in vivo experiments, we confirmed the immunocidal effect of Dio-potentiated suicide gene therapy on melanoma. The results showed that Dio upregulated connexin 43 (Cx43) expression and improved gap junction intercellular communication (GJIC) in B16 cells while increasing the cross-presentation of antigens by dendritic cells (DCs), eventually promoting the activation and antitumor immune killing effects of CD8+ T lymphocytes. In contrast, inhibition or blockade of the GJIC function (overexpression of mutant Cx43 tumor cells/Gap26) partially reversed the potentiating effect. The significant synergistic effect of Dio on HSV-Tk/GCV suicide gene therapy was further investigated in a B16 xenograft mouse model. The increased number and activation ratio of CD8+ T lymphocytes and the levels of Gzms-B, IFN-γ, and TNF-α in mice reconfirmed the potential modulatory effects of Dio on the immune system. Taken together, Dio targets Cx43 to enhance GJIC function, improve the antigens cross-presentation of DCs, and activate the antitumor immune effect of CD8+ T lymphocytes, thereby providing insights into the potential immunomodulatory mechanism underlying combination therapy.

Gap junction intercellular communication (GJIC) plays an important role in cell growth, development and homeostasis. Connexin 43 (Cx43) is an important half-channel protein responsible for gap junction formation. Platelet-derived growth factor AA (PDGF-AA) regulates the proliferation, migration, metabolism, apoptosis and cell cycle of chondrocytes. However, the role of PDGF-AA in gap junction intercellular communication in chondrocytes is not fully understood. In the current study, we performed experiments to explore the effect of PDGF-AA on GJIC and its underlying biomechanical mechanism.
qPCR was performed to determine the expression of PDGF, PDGFR and connexin family genes in chondrocytes and/or cartilage. A scrape loading/dye transfer assay was used to determine GJIC. Western blot analysis was applied to detect the expression of Cx43 and PI3K/Akt signaling pathway proteins. Immunofluorescence staining was utilized to examine protein distribution. Scanning electron microscopy was used to delineate the morphology of chondrocytes.
Expression of PDGF-A mRNA was highest among the PDGF family in chondrocytes and cartilage tissues. PDGF-AA promoted functional GJIC formation in chondrocytes by upregulating the expression of Cx43. Enhanced functional GJIC formation in chondrocytes induced by PDGF-AA occurred through the activation of PI3K/Akt signaling and its nuclear accumulation.
For the first time, this study provides evidence demonstrating the role of PDGF-AA in cell-to-cell communication in chondrocytes through mediating Cx43 expression.