Mechano-activated connexin hemichannels and glutathione transport protect lens fiber cells against oxidative insults.

Long-lived lens fiber cells require a robust cellular protective function against oxidative insults to maintain their hemostasis and viability; however, the underlying mechanism is largely obscure. In this study, we unveiled a new mechanism that protects lens fiber cells against oxidative stress-induced cell death. We found that mechano-activated connexin (Cx) hemichannels (HCs) mediate the transport of glutathione (GSH) into chick embryonic fibroblasts (CEF) and primary lens fiber cells, resulting in a decrease in the accumulation of intracellular reactive oxygen species induced by both H2O2 and ultraviolet B, providing protection to lens fiber cells against cell apoptosis and necrosis. Furthermore, HCs formed by both homomeric Cx50 or Cx46 and heteromeric Cx50/Cx46 were mechanosensitive and could transport GSH into CEF cells. Notably, mechano-activated Cx50 HCs exhibited a greater capacity to transport GSH than Cx46 HCs. Consistently, the deficiency of Cx50 in single lens fiber cells led to a higher level of oxidative stress. Additionally, outer cortical short lens fiber cells expressing full length Cxs demonstrated greater resistance to oxidative injury compared to central core long lens fibers. Taken together, our results suggest that the activation of Cx HCs by interstitial fluid flow in cultured epithelial cells and isolated fiber cells shows that HCs can serve as a pathway for moving GSH across the cell membrane to offer protection against oxidative stress.

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.

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.

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.

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).

Osteocytic Connexin Hemichannels Modulate Oxidative Bone Microenvironment and Breast Cancer Growth.

Osteocytes, the most abundant bone cell types embedded in the mineral matrix, express connexin 43 (Cx43) hemichannels that play important roles in bone remodeling and osteocyte survival. Estrogen deficiency decreases osteocytic Cx43 hemichannel activity and causes a loss in osteocytes' resistance to oxidative stress (OS). In this study, we showed that OS reduced the growth of both human (MDA-MB-231) and murine (Py8119) breast cancer cells. However, co-culturing these cells with osteocytes reduced the inhibitory effect of OS on breast cancer cells, and this effect was ablated by the inhibition of Cx43 hemichannels. Py8119 cells were intratibially implanted in the bone marrow of ovariectomized (OVX) mice to determine the role of osteocytic Cx43 hemichannels in breast cancer bone metastasis in response to OS. Two transgenic mice overexpressing dominant-negative Cx43 mutants, R76W and Δ130-136, were adopted for this study; the former inhibits gap junctions while the latter inhibits gap junctions and hemichannels. Under normal conditions, Δ130-136 mice had significantly more tumor growth in bone than that in WT and R76W mice. OVX increased tumor growth in R76W but had no significant effect on WT mice. In contrast, OVX reduced tumor growth in Δ130-136 mice. To confirm the role of OS, WT and Δ130-136 mice were administered the antioxidant N-acetyl cysteine (NAC). NAC increased tumor burden and growth in Δ130-136 mice but not in WT mice. Together, the data suggest that osteocytes and Cx43 hemichannels play pivotal roles in modulating the oxidative microenvironment and breast cancer growth in the bone.

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.

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.

Mechanosensitive collaboration between integrins and connexins allows nutrient and antioxidant transport into the lens.

The delivery of glucose and antioxidants is vital to maintain homeostasis and lens transparency. Here, we report a new mechanism whereby mechanically activated connexin (Cx) hemichannels serve as a transport portal for delivering glucose and glutathione (GSH). Integrin α6ß1 in outer cortical lens fiber activated by fluid flow shear stress (FFSS) induced opening of hemichannels. Inhibition of α6 activation prevented hemichannel opening as well as glucose and GSH uptake. The activation of integrin ß1, a heterodimeric partner of α6 in the absence of FFSS, increased Cx50 hemichannel opening. Hemichannel activation by FFSS depended on the interaction of integrin α6 and Cx50 C-terminal domain. Moreover, hemichannels in nuclear fiber were unresponsive owing to Cx50 truncation. Taken together, these results show that mechanically activated α6ß1 integrin in outer cortical lens fibers leads to opening of hemichannels, which transport glucose and GSH into cortical lens fibers. This study unveils a new transport mechanism that maintains metabolic and antioxidative function of the lens.

Effects of Interleukin-6 on posterior capsular opacification.

The purpose of this work was to determine the effects of interleukin-6 (IL-6) on the development of posterior capsular opacification (PCO) in vitro and in vivo. Western blot and real-time PCR were used to test the IL-6-induced epithelial-mesenchymal transition (EMT) marker α-smooth muscle actin (α-SMA), the extracellular matrix (ECM) markers fibronectin (Fn) and type I collagen (COL-1), transforming growth factor ß2 (TGF-ß2), and the activation and role of the JAK/STAT3 signaling pathway in human lens epithelial cells (HLECs). Immunocytofluorescence staining was performed to detect gp130 and IL-6Rα expression in HLECs. Rat PCO models were then established to examine the impact of STAT3 knockdown by shRNA adeno-associated virus on PCO development, and immunohistochemical staining was performed to detect the expression of Fn in the anterior and posterior capsule in vivo. We found that IL-6 promotes the expression of Fn, COL-1, TGF-ß2, p-JAK2 and p-STAT3 in HLECs but exerts little effect on α-SMA. The JAK/STAT3 inhibitor WP1066 effectively suppressed the IL-6-induced expression of Fn and COL-1 in lens epithelial cells. STAT3 knockdown effectively inhibited the development of PCO in rats and significantly reduced the expression of Fn in the anterior and posterior capsule. These data suggest that IL-6 contributes to the development of PCO by promoting TGF-ß2 activation and ECM synthesis through a JAK/STAT3 signaling-dependent mechanism. Furthermore, inhibiting JAK/STAT3 signaling effectively impairs both PCO development in rats and ECM synthesis in the lens capsule.