Evaluation of Connexin Hemichannel Activity In Vivo.

Connexin hemichannels (Cx HCs) are hexameric structures at the cell plasma membrane, whose function as membrane transport proteins allows for the passive flow of small hydrophilic molecules and ions (≤1 kDa) between the cytosol and the extracellular environment. Activation of Cx HCs is highly dependent on pathological conditions. HC activity provokes changes in the microenvironment, inducing the dissemination of signaling molecules in both an autocrine and paracrine manner. Given the elicitation of a variety of signaling pathways, and assortment of Cx species and dispersion throughout the body, Cx HCs have been implicated in a range of processes such as cell proliferation, differentiation, cell death, and tissue modeling and remodeling. While studying the expression and localization of Cx HCs can be done using traditional laboratory techniques, such as immunoblot analysis, measuring the functionality/activity of the HCs requires a more explicit methodology and is essential for determining Cx-mediated physiological changes. The study of Cx HC function/activity has focused mainly on in vitro measurements through electrophysiological characterization or, more commonly, using HC-permeable dye uptake studies. Here, we describe the use of dye uptake to measure Cx HC activity in vivo using mechanically stimulated osteocytic Cx43 HCs with Evans blue dye as our model.

Protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high-titer retroviral RCAS(A) infection.

Connexins (Cxs) play a crucial role in maintaining lens transparency. Here, we present a protocol for altering Cx hemichannel (HC) function in primary chicken lens fiber cells using high-titer retroviral replication competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor (A) infection. We describe steps for incubating eggs, isolating lenses, culturing cells, preparing reagents, and infecting cells. We then detail cell treatment and detection of apoptosis and death. This protocol can assess protein kinase A, HC activity, and increased glutathione transport for protecting lens fiber cells against oxidative stress. For complete details on the use and execution of this protocol, please refer to Liu et al.,1 Riquelme et al.,2 Shi et al.,3 Jiang,4 and Rath et al.5.

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens.

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

Protein kinase A activation alleviates cataract formation via increased gap junction intercellular communication.

Cataract is the leading cause of blindness worldwide. Here, we reported a potential, effective therapeutic mean for cataract prevention and treatment. Gap junction communication, an important mechanism in maintaining lens transparency, is increased by protein kinase A (PKA). We found that PKA activation reduced cataracts induced by oxidative stress, increased gap junctions/hemichannels in connexin (Cx) 50, Cx46 or Cx50 and Cx46 co-expressing cells, and decreased reactive oxygen species (ROS) levels. However, ROS reduction was shown in wild-type, Cx46 and Cx50 knockout, but not in Cx46/Cx50 double KO lens. In addition, PKA activation protects lens fiber cell death induced by oxidative stress via hemichannel-mediated glutathione transport. Connexin deletion increased lens opacity induced by oxidative stress associated with reduction of anti-oxidative stress gene expression. Together, our results suggest that PKA activation through increased connexin channels in lens fiber cell decreases ROS levels and cell death, leading to alleviated cataracts.

The second extracellular domain of connexin 50 is important for in cell adhesion, lens differentiation, and adhesion molecule expression.

Connexin (Cx)-forming channels play essential roles in maintaining lens homeostasis and transparency. We showed here channel-independent roles of Cx50 in cell-cell adhesion and confirmed the second extracellular (E2) domain as a critical domain for cell adhesion function. We found that cell adhesion decreased in cells expressing chimeric Cx50 in which the E2 domain was swapped with the E2 domain of either Cx43 or Cx46. In contrast, adhesion increased in cells expressing chimeric Cx43 and Cx46 with the Cx50 (E2) domain. This function is Cx channel-independent and Cx50 E2 domain-dependent cell adhesion acting in both homotypic and heterotypic manners. In addition, we generated eight site mutations of unique residues between Cx50 and the other two lens Cxs and found that mutation of any one of the residues abolished the adhesive function. Moreover, expression of adhesive-impaired mutants decreased adhesion-related proteins, N-cadherin and ß-catenin. Expression of the adhesion-impaired Cx50W188P mutant in embryonic chick lens caused enlarged extracellular spaces, distorted fiber organization, delayed nuclear condensation, and cortical cataracts. In summary, the results from both in vitro and in vivo studies demonstrate the importance of the adhesive function of Cx50 in the lens.

Mechanosensitive piezo1 calcium channel activates connexin 43 hemichannels through PI3K signaling pathway in bone.

BACKGROUND: Mechanical loading promotes bone formation and osteocytes are a major mechanosensory cell in the bone. Both Piezo1 channels and connexin 43 hemichannels (Cx43 HCs) in osteocytes are important players in mechanotransduction and anabolic function by mechanical loading. However, the mechanism underlying mechanotransduction involving Piezo1 channels and Cx43 HCs in osteocytes and bone remains unknown. RESULTS: We showed that, like mechanical loading, Piezo1 specific agonist Yoda1 was able to increase intracellular Ca2+ signaling and activate Cx43 HCs, while Yoda1 antagonist Dooku1 inhibited Ca2+ and Cx43 HC activation induced by both mechanical loading and Yoda1. Moreover, the intracellular Ca2+ signal activated by Yoda1 was reduced by the inhibition of Cx43 HCs and pannexin1 (Panx1) channels, as well as ATP-P2X receptor signaling. Piezo1 and Cx43 HCs were co-localized on the osteocyte cell surface, and Yoda1-activated PI3K-Akt signaling regulated the opening of Cx43 HCs. Furthermore, Cx43 HCs opening by mechanical loading on tibias was ablated by inhibition of Piezo1 activation in vivo. CONCLUSION: We demonstrated that upon mechanical stress, increased intracellular Ca2+ activated by Piezo1 regulates the opening of HCs through PI3K-Akt and opened Cx43 HCs, along with Panx1 channels, and ATP-P2X signaling sustain the intracellular Ca2+ signal, leading to bone anabolic function.

Connexin 43 hemichannels regulate mitochondrial ATP generation, mobilization, and mitochondrial homeostasis against oxidative stress.

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.

Osteocytes regulate bone anabolic response to mechanical loading in male mice via activation of integrin α5.

Physical mechanical stimulation can maintain and even increase bone mass. Here, we report an important role of osteocytic integrin α5 in regulating the anabolic response of bone to mechanical loading using an Itga5 conditional gene knockout (cKO) mouse model. Integrin α5 gene deletion increased apoptotic osteocytes and reduced cortical anabolic responses to tibial compression including decreased endosteal osteoblasts and bone formation, and increased endosteal osteoclasts and bone resorption, contributing to the decreased bone area fraction and biomechanical properties, leading to an enlarged bone marrow area in cKO mice. Similar disruption of anabolic responses to mechanical loading was also detected in cKO trabecular bone. Moreover, integrin α5 deficiency impeded load-induced Cx43 hemichannel opening, and production and release of PGE2, an anabolic factor, resulting in attenuated effects of the loading on catabolic sclerostin (SOST) reduction and anabolic ß-catenin increase. Together, this study shows an indispensable role of integrin α5 in osteocytes in the anabolic action of mechanical loading on skeletal tissue through activation of hemichannels and PGE2-evoked gene expression. Integrin α5 could act as a potential new therapeutic target for bone loss, especially in the elderly population with impeded mechanical sensitivity.

Connexin 43 Hemichannels Regulate Osteoblast to Osteocyte Differentiation.

Connexin 43 (Cx43) is the predominant connexin subtype expressed in osteocytes. Osteocytes, accounting for 90%-95% of total bone cells, function as orchestrators coordinating balanced activity between bone-resorbing osteoclasts and bone-forming osteoblasts. In this study, two newly developed osteocytic cell lines, OCY454 and IDG-SW3, were used to determine the role of Cx43 gap junctions and hemichannels (HCs) in the regulation of osteoblast to osteocyte differentiation. We found that the Cx43 level was substantially increased during the differentiation of IDG-SW3 cells and is also much higher than that of OCY454 cells. We knocked down Cx43 expression using the lentiviral CRISPR/Cas9 approach and inhibition of Cx43 HCs using Cx43 (E2) antibody in IDG-SW3 cells. Cx43 knockdown (KD) or Cx43 HC inhibition decreased gene expression for osteoblast and osteocyte markers, including alkaline phosphatase, type I collagen, dentin matrix protein 1, sclerostin, and fibroblast growth factor 23, whereas increasing the osteoclastogenesis indicator and the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio at early and late differentiation stages. Moreover, mineralization was remarkably attenuated in differentiated Cx43-deficient IDG-SW3 cells compared to ROSA26 control. The conditioned medium collected from fully differentiated IDG-SW3 cells with Cx43 KD promoted osteoclastogenesis of RAW264.7 osteoclast precursors. Our results demonstrated that Cx43 HCs play critical roles in osteoblast to osteocyte differentiation process and regulate osteoclast differentiation via secreted factors.

Beyond the Channels: Adhesion Functions of Aquaporin 0 and Connexin 50 in Lens Development.

Lens, an avascular tissue involved in light transmission, generates an internal microcirculatory system to promote ion and fluid circulation, thus providing nutrients to internal lens cells and excreting the waste. This unique system makes up for the lack of vasculature and distinctively maintains lens homeostasis and lens fiber cell survival through channels of connexins and other transporters. Aquaporins (AQP) and connexins (Cx) comprise the majority of channels in the lens microcirculation system and are, thus, essential for lens development and transparency. Mutations of AQPs and Cxs result in abnormal channel function and cataract formation. Interestingly, in the last decade or so, increasing evidence has emerged suggesting that in addition to their well-established channel functions, AQP0 and Cx50 play pivotal roles through channel-independent actions in lens development and transparency. Specifically, AQP0 and Cx50 have been shown to have a unique cell adhesion function that mediates lens development and transparency. Precise regulation of cell-matrix and cell-cell adhesion is necessary for cell migration, a critical process during lens development. This review will provide recent advances in basic research of cell adhesion mediated by AQP0 and Cx50.

Connexin hemichannels with prostaglandin release in anabolic function of bone to mechanical loading.

Mechanical stimulation, such as physical exercise, is essential for bone formation and health. Here, we demonstrate the critical role of osteocytic Cx43 hemichannels in anabolic function of bone in response to mechanical loading. Two transgenic mouse models, R76W and Δ130-136, expressing dominant-negative Cx43 mutants in osteocytes were adopted. Mechanical loading of tibial bone increased cortical bone mass and mechanical properties in wild-type and gap junction-impaired R76W mice through increased PGE2, endosteal osteoblast activity, and decreased sclerostin. These anabolic responses were impeded in gap junction/hemichannel-impaired Δ130-136 mice and accompanied by increased endosteal osteoclast activity. Specific inhibition of Cx43 hemichannels by Cx43(M1) antibody suppressed PGE2 secretion and impeded loading-induced endosteal osteoblast activity, bone formation and anabolic gene expression. PGE2 administration rescued the osteogenic response to mechanical loading impeded by impaired hemichannels. Together, osteocytic Cx43 hemichannels could be a potential new therapeutic target for treating bone loss and osteoporosis.

Studying macrophage activation in immune-privileged lens through CSF-1 protein intravitreal injection in mouse model.

Macrophage (MΦ) activation and promotion of fibrosis are critical processes in lens capsule healing after injury. Here, we detail a protocol that induces MΦ2 formation within the vitreous body of the eye. Our procedure combines the use of an intravitreal injection of a growth factor (CSF-1) and immunofluorescence to confirm the presence of MΦ2 and fibrotic tissue formation. This protocol allows assessment of the distribution of macrophages and quantification of fibrotic tissue formation/sealing within the vitreous body of mouse eyes. For complete details on the use and execution of this profile, please refer to Li et al. (2021), Gerhardt et al. (2003), Kubota et al. (2009).

Data on Connexin 43 hemichannels regulation of cellular redox in lens.

This article describes a dataset that is related to the research paper "Connexin hemichannels regulate redox potential via metabolite exchange and protect lens against cellular oxidative damage". Growing evidence demonstrates that oxidative stress is a key event in cataract formation. Hemichannels (HCs) formed by Connexin (Cx) 43, a Cx subtype only present in the epithelium of lens tissue, mediate the exchange of small molecules between the intracellular and extracellular environments, including redox-related metabolic molecules, such as glutathione (GSH) and reactive oxygen species (ROS). Here, we used a Cx43 heterozygous mouse model, Cx43E2 antibody (a specific Cx43 HC blocker), and knocked down Cx43 expression by siRNA in human lens epithelial HLE-B3 cells to assess the oxidative response of Cx43 HCs to H2O2 and UVB radiation. Western blot analysis of heterozygous Cx43-null (Cx43+/-) mouse lenses showed the haploinsufficiency of Cx43 protein. We further assessed anti-oxidative gene expression in response to H2O2 and UVB radiation treatment in the Cx43-deficient lens epithelial cells. This dataset will be useful for understanding the critical role of Cx43 HCs in maintaining redox homeostasis in the lens under oxidative stress.

ATP Inhibits Breast Cancer Migration and Bone Metastasis through Down-Regulation of CXCR4 and Purinergic Receptor P2Y11.

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 Gap Junctions and Hemichannels in Modulating Lens Redox Homeostasis and Oxidative Stress in Cataractogenesis.

The lens is continuously exposed to oxidative stress insults, such as ultraviolet radiation and other oxidative factors, during the aging process. The lens possesses powerful oxidative stress defense systems to maintain its redox homeostasis, one of which employs connexin channels. Connexins are a family of proteins that form: (1) Hemichannels that mediate the communication between the intracellular and extracellular environments, and (2) gap junction channels that mediate cell-cell communication between adjacent cells. The avascular lens transports nutrition and metabolites through an extensive network of connexin channels, which allows the passage of small molecules, including antioxidants and oxidized wastes. Oxidative stress-induced post-translational modifications of connexins, in turn, regulates gap junction and hemichannel permeability. Recent evidence suggests that dysfunction of connexins gap junction channels and hemichannels may induce cataract formation through impaired redox homeostasis. Here, we review the recent advances in the knowledge of connexin channels in lens redox homeostasis and their response to cataract-related oxidative stress by discussing two major aspects: (1) The role of lens connexins and channels in oxidative stress and cataractogenesis, and (2) the impact and underlying mechanism of oxidative stress in regulating connexin channels.

Connexin hemichannels regulate redox potential via metabolite exchange and protect lens against cellular oxidative damage.

Increased oxidative stress contributes to cataract formation during aging. Anterior epithelial cells are a frontline antioxidant defense system with powerful capacities to maintain redox homeostasis and lens transparency. In this study, we report a new molecular mechanism of connexin (Cx) hemichannels (HCs) in lens epithelial cells to protect lens against oxidative stress. Our results showed haploinsufficiency of Cx43 elevated oxidative stress and susceptibility to cataracts in the mouse lens. Cx43 HCs opened in response to hydrogen peroxide (H2O2) or ultraviolet radiation (UVR) in human lens epithelium HLE-B3 cells, and this activation contributed to a cellular protective mechanism against oxidative stress-induced apoptotic cell death. Furthermore, we found that Cx43 HCs mediated the exchange of oxidants and antioxidants in lens epithelial cells undergoing oxidative stress. These transporting activities facilitated a reduction of intracellular reactive oxygen species (ROS) accumulation and maintained the intracellular glutathione (GSH) level through the exchange of redox metabolites and change of anti-oxidative gene expression. In addition, we show that Cx43 HCs can be regulated by the intracellular redox state and this regulation is mediated by residue Cys260 located at the Cx43 C-terminus. Together, our results demonstrate that Cx43 HCs activated by oxidative stress in the lens epithelial cells play a key role in maintaining redox homeostasis in lens under oxidative stress. Our findings contribute to advancing our understanding of oxidative stress induced lens disorders, such as age-related non-congenital cataracts.

Macrophage recruitment in immune-privileged lens during capsule repair, necrotic fiber removal, and fibrosis.

Emerging evidence challenges the lens as an immune-privileged organ. Here, we provide a direct mechanism supporting a role of macrophages in lens capsule rupture repair. Posterior lens capsule rupture in a connexin 50 and aquaporin 0 double-knockout mouse model resulted in lens tissue extrusion into the vitreous cavity with formation of a "tail-like" tissue containing delayed regressed hyaloid vessels, fibrotic tissue and macrophages at postnatal (P) 15 days. The macrophages declined after P 30 days with M2 macrophages detected inside the lens. By P 90 days, the "tail-like" tissue completely disappeared and the posterior capsule rupture was sealed with thick fibrotic tissue. Colony-stimulating factor 1 (CSF-1) accelerated capsule repair, whereas inhibition of the CSF-1 receptor delayed the repair. Together, these results suggest that lens posterior rupture leads to the recruitment of macrophages delivered by the regression delayed hyaloid vessels. CSF-1-activated M2 macrophages mediate capsule rupture repair and development of fibrosis.

Development of a potent embryonic chick lens model for studying congenital cataracts in vivo.

Congenital cataracts are associated with gene mutations, yet the underlying mechanism remains largely unknown. Here we reported an embryonic chick lens model that closely recapitulates the process of cataract formation. We adopted dominant-negative site mutations that cause congenital cataracts, connexin, Cx50E48K, aquaporin 0, AQP0R33C, αA-crystallin, CRYAA R12C and R54C. The recombinant retroviruses containing these mutants were microinjected into the occlusive lumen of chick lenses at early embryonic development. Cx50E48K expression developed cataracts associated with disorganized nuclei and enlarged extracellular spaces. Expression of AQP0R33C resulted in cortical cataracts, enlarged extracellular spaces and distorted fiber cell organization. αA crystallin mutations distorted lens light transmission and increased crystalline protein aggregation. Together, retroviral expression of congenital mutant genes in embryonic chick lenses closely mimics characteristics of human congenital cataracts. This model will provide an effective, reliable in vivo system to investigate the development and underlying mechanism of cataracts and other genetic diseases.

Inhibition of astrocyte hemichannel improves recovery from spinal cord injury.

Spinal cord injury (SCI) causes severe disability, and the current inability to restore function to the damaged spinal cord leads to lasting detrimental consequences to patients. One strategy to reduce SCI morbidity involves limiting the spread of secondary damage after injury. Previous studies have shown that connexin 43 (Cx43), a gap junction protein richly expressed in spinal cord astrocytes, is a potential mediator of secondary damage. Here, we developed a specific inhibitory antibody, mouse-human chimeric MHC1 antibody (MHC1), that inhibited Cx43 hemichannels, but not gap junctions, and reduced secondary damage in 2 incomplete SCI mouse models. MHC1 inhibited the activation of Cx43 hemichannels in both primary spinal astrocytes and astrocytes in situ. In both SCI mouse models, administration of MHC1 after SCI significantly improved hind limb locomotion function. Remarkably, a single administration of MHC1 30 minutes after injury improved the recovery up to 8 weeks post-SCI. Moreover, MHC1 treatment decreased gliosis and lesion sizes, increased white and gray matter sparing, and improved neuronal survival. Together, these results suggest that inhibition of Cx43 hemichannel function after traumatic SCI reduces secondary damage, limits perilesional gliosis, and improves functional recovery. By targeting hemichannels specifically with an antibody, this study provides a potentially new, innovative therapeutic approach in treating SCI.

Mechanotransduction via the coordinated actions of integrins, PI3K signaling and Connexin hemichannels.

Mechanical loading opens connexin 43 (Cx43) hemichannels (HCs), leading to the release of bone anabolic molecules, such as prostaglandins, from mechanosensitive osteocytes, which is essential for bone formation and remodeling. However, the mechanotransduction mechanism that activates HCs remains elusive. Here, we report a unique pathway by which mechanical signals are effectively transferred between integrin molecules located in different regions of the cell, resulting in HC activation. Both integrin α5 and αV were activated upon mechanical stimulation via either fluid dropping or flow shear stress (FSS). Inhibition of integrin αV activation or ablation of integrin α5 prevented HC opening on the cell body when dendrites were mechanically stimulated, suggesting mechanical transmission from the dendritic integrin αV to α5 in the cell body during HC activation. In addition, HC function was compromised in vivo, as determined by utilizing an antibody blocking αV activation and α5-deficient osteocyte-specific knockout mice. Furthermore, inhibition of integrin αV activation, but not that of α5, attenuated activation of the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway upon mechanical loading, and the inhibition of PI3K/AKT activation blocked integrin α5 activation and HC opening. Moreover, HC opening was blocked only by an anti-integrin αV antibody at low but not high FSS levels, suggesting that dendritic αV is a more sensitive mechanosensor than α5 for activating HCs. Together, these results reveal a new molecular mechanism of mechanotransduction involving the coordinated actions of integrins and PI3K/AKT in osteocytic dendritic processes and cell bodies that leads to HC opening and the release of key bone anabolic factors.