Oxidative stress is a major risk factor that causes osteocyte cell death and bone loss. Prior studies primarily focus on the function of cell surface expressed Cx43 channels. Here, we reported a new role of mitochondrial Cx43 (mtCx43) and hemichannels (HCs) in modulating mitochondria homeostasis and function in bone osteocytes under oxidative stress. In murine long bone osteocyte-Y4 cells, the translocation of Cx43 to mitochondria was increased under H₂O₂-induced oxidative stress. H₂O₂ increased the mtCx43 level accompanied by elevated mtCx43 HC activity, determined by dye uptake assay. Cx43 knockdown (KD) by the CRISPR-Cas9 lentivirus system resulted in impairment of mitochondrial function, primarily manifested as decreased ATP production. Cx43 KD had reduced intracellular reactive oxidative species levels and mitochondrial membrane potential. Additionally, live-cell imaging results demonstrated that the proton flux was dependent on mtCx43 HCs because its activity was specifically inhibited by an antibody targeting Cx43 C-terminus. The co-localization and interaction of mtCx43 and ATP synthase subunit F (ATP5J2) were confirmed by Förster resonance energy transfer and a protein pull-down assay. Together, our study suggests that mtCx43 HCs regulate mitochondrial ATP generation by mediating K⁺, H⁺, and ATP transfer across the mitochondrial inner membrane and the interaction with mitochondrial ATP synthase, contributing to the maintenance of mitochondrial redox levels in response to oxidative stress.

ATP released by bone osteocytes is shown to activate purinergic signaling and inhibit the metastasis of breast cancer cells into the bone. However, the underlying molecular mechanism is not well understood. Here, we demonstrate the important roles of the CXCR4 and P2Y11 purinergic receptors in mediating the inhibitory effect of ATP on breast cancer cell migration and bone metastasis. Wound-healing and transwell migration assays showed that non-hydrolysable ATP analogue, ATPγS, inhibited migration of bone-tropic human breast cancer cells in a dose-dependent manner. BzATP, an agonist for P2X7 and an inducer for P2Y11 internalization, had a similar dose-dependent inhibition on cell migration. Both ATPγS and BzATP suppressed the expression of CXCR4, a chemokine receptor known to promote breast cancer bone metastasis, and knocking down CXCR4 expression by siRNA attenuated the inhibitory effect of ATPγS on cancer cell migration. While a P2X7 antagonist A804598 had no effect on the impact of ATPγS on cell migration, antagonizing P2Y11 by NF157 ablated the effect of ATPγS. Moreover, the reduction in P2Y11 expression by siRNA decreased cancer cell migration and abolished the impact of ATPγS on cell migration and CXCR4 expression. Similar to the effect of ATPγS on cell migration, antagonizing P2Y11 inhibited bone-tropic breast cancer cell migration in a dose-dependent manner. An in vivo study using an intratibial bone metastatic model showed that ATPγS inhibited breast cancer growth in the bone. Taken together, these results suggest that ATP inhibits bone-tropic breast cancer cells by down-regulating the P2Y11 purinergic receptor and the down-regulation of CXCR4 expression.

Connexin 43 (Cx43) protein forms hemichannels (connexons) and gap junctions, with hemichannels consisting of six Cx43 molecules and gap junctions formed by two hemichannels. While gap junctions are prevalent in organs like the heart and liver, hemichannels are found in specific cell types, such as astrocytes and osteocytes. They allow the passage of small molecules (<1.5 kDa) between the cytoplasm and extracellular matrix. Cx43 hemichannels have emerged as potential therapeutic targets in various diseases, including central nervous system disorders, bone-related diseases, diabetic complications, wound healing, and cancers. Aberrant hemichannel opening can worsen conditions by releasing inflammatory elements, such as causing gliosis in neuronal cells. Conversely, functional hemichannels may inhibit cancer cell growth and metastasis. Recent studies are revealing new mechanisms of Cx43 hemichannels, broadening their therapeutic applications and highlighting the importance of regulating their activity for improved disease outcomes.

Bone–muscle crosstalk plays an important role in skeletal biomechanical function, the progression of numerous pathological conditions, and the modulation of local and distant cellular environments. Previous work has revealed that the deletion of connexin (Cx) 43 in osteoblasts, and consequently, osteocytes, indirectly compromises skeletal muscle formation and function. However, the respective roles of Cx43-formed gap junction channels (GJs) and hemichannels (HCs) in the bone–muscle crosstalk are poorly understood. To this end, we used two Cx43 osteocyte-specific transgenic mouse models expressing dominant negative mutants, Δ130–136 (GJs and HCs functions are inhibited), and R76W (only GJs function is blocked), to determine the effect of these two types of Cx43 channels on neighboring skeletal muscle. Blockage of osteocyte Cx43 GJs and HCs in Δ130–136 mice decreased fast-twitch muscle mass with reduced muscle protein synthesis and increased muscle protein degradation. Both R76W and Δ130–136 mice exhibited decreased muscle contractile force accompanied by a fast-to-slow fiber transition in typically fast-twitch muscles. In vitro results further showed that myotube formation of C2C12 myoblasts was inhibited after treatment with the primary osteocyte conditioned media (PO CM) from R76W and Δ130–136 mice. Additionally, prostaglandin E2 (PGE2) level was significantly reduced in both the circulation and PO CM of the transgenic mice. Interestingly, the injection of PGE2 to the transgenic mice rescued fast-twitch muscle mass and function; however, this had little effect on protein synthesis and degradation. These findings indicate a channel-specific response: inhibition of osteocytic Cx43 HCs decreases fast-twitch skeletal muscle mass alongside reduced protein synthesis and increased protein degradation. In contrast, blockage of Cx43 GJs results in decreased fast-twitch skeletal muscle contractile force and myogenesis, with PGE2 partially accounting for the measured differences.

Bone is a dynamic organ constantly undergoing remodeling with both bone formation and resorption. Bone formation is mediated by osteoblasts originating from the differentiation of bone marrow (BM) mesenchymal stem and progenitor cells (BM-MSPCs). However, how bone cells communicate with BM-MSPCs to coordinate bone formation remains largely elusive. Here, we unveil a key role of osteocyte connexin 43 (Cx43) hemichannels in regulating the lineage commitment of BM-MSPCs. Two transgenic mouse models expressing dominant negative Cx43 mutants in osteocytes were used: R76W (inhibiting gap junctions) and Δ130 to 136 (inhibiting both hemichannels and gap junctions). BM-MSPCs from Δ130 to 136 mice showed enhanced adipogenic differentiation and reduced osteogenic potential, leading to increased BM adipocytes. Flow cytometry and single-cell RNA sequencing revealed shifts in BM-MSPC subsets, less osteogenic-biased MSPCs, and more adipogenic-biased MSPCs in Δ130 to 136 mice. Conversely, R76W, with more functional hemichannels, exhibited effects similar to WT mice or even greater opposite effects than Δ130 to 136 mice. Prostaglandin E ₂ (PGE ₂ ), released from active Cx43 hemichannels, inhibited adipogenesis and promoted osteogenesis via the PGE ₂ receptor EP4 and ERK1/2 signaling. Inhibition of Cx43 hemichannels or EP4 led to increased adipogenic-biased MSPCs. Moreover, administration of a Cx43(M1) antibody, which inhibits hemichannels, substantially increased BM adipocytes, accompanied by increased adipogenic-biased MSPCs, and decreased osteogenic-biased MSPCs. Our study highlights the pivotal role of osteocyte Cx43 hemichannels in BM-MSPC fate decision through PGE ₂ release, providing insights into the precise and highly regulated communication between matrix-bound bone cells and BM-MSPCs, which dictates bone formation and remodeling.

Fibrosis initially appears as a normal response to damage, where activated fibroblasts produce large amounts of the extracellular matrix (ECM) during the wound healing process to assist in the repair of injured tissue. However, the excessive accumulation of the ECM, unresolved by remodeling mechanisms, leads to organ dysfunction. Connexins, a family of transmembrane channel proteins, are widely recognized for their major roles in fibrosis, the epithelial–mesenchymal transition (EMT), and wound healing. Efforts have been made in recent years to identify novel mediators and targets for this regulation. Connexins form gap junctions and hemichannels, mediating communications between neighboring cells and inside and outside of cells, respectively. Recent evidence suggests that connexins, beyond forming channels, possess channel-independent functions in fibrosis, the EMT, and wound healing. One crucial channel-independent function is their role as the primary functional component for cell adhesion. Other channel-independent functions of connexins involve their roles in mitochondria and exosomes. This review summarizes the latest advances in the channel-dependent and independent roles of connexins in fibrosis, the EMT, and wound healing, with a particular focus on eye diseases, emphasizing their potential as novel, promising therapeutic targets.