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.

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.

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.

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.