Project Summary Lens, an avascular organ, relies heavily on a network of transporting systems to deliver nutrients and other essential components to bulky lens fibers and excrete wastes. Differentiated nuclear fiber cells never turn over, and oxidative stress is a major cause of age-related cataracts. Fluid flow associated with lens microcirculation is thought to help deliver nutrients, antioxidants, and small molecules to inner lens fibers. Connexin (Cx)- forming gap junction channels (GJs) play an essential role in the metabolic homeostasis of the lens. Besides GJs, connexins form hemichannels (HCs), permitting the transport of molecules between the cell and its extracellular environment. Our preliminary studies indicate that lens epithelial HCs are activated by FSS and release GSH and ATP; these released factors protect lens fiber cells against oxidative stress and promote lens fiber differentiation, respectively. As such, we hypothesize that (1) The epithelial HCs formed by Cx43 and/or Cx50 activated by FSS regulate GSH de novo synthesis, release, and redox hemostasis; (2) GSH released by epithelial HCs is taken up by HCs in cortical fiber cells and transferred to nuclear fiber cells to maintain lens fiber cell redox homeostasis and viability; (3) Epithelial HCs activated by FSS generate secretomes and released ATP, and its binding to purinergic P2X receptors (P2XRs) (i.e., P2XR3 or/and P2XR7) regulate Ca2+ signaling and fiber cell differentiation. The goal is to elucidate the roles of lens epithelial HCs activated under a physiological level of mechanical stimulation in hemostasis, differentiation, and protection against oxidative insults in lens fiber cells. In this proposal, first, we will determine if Cx HCs activated by FSS serve as a major pathway in regulating GSH synthesis, export, and redox homeostasis in lens epithelial cells. Second, we will test if GSH released by lens epithelial HCs is taken up by mechano-activated HCs in cortical lens fibers and transferred to nuclear fibers via GJs functions in reducing ROS and protecting fiber cells against oxidative stress. Third, we will determine if ATP released by lens epithelial HCs and activation of purinergic receptor signaling regulates fiber cell differentiation and globally investigate secretomes by FSS-activated epithelial HCs. One of the major innovative aspects is that this proposal aims to uncover novel, integrated roles of Cx HCs in lens epithelial and fiber cells and GJs in antioxidant transport, fiber cell protection, and the role of ATP released by epithelial HCs and activated P2XRs in lens fiber differentiation. We will use chick primary lens cultures, chick lens in situ, ex vivo mouse lens explants of wild-type and knockout mice, HC-specific inhibiting antibodies, dominant mutants, and mechanical loading models. It is our expectation that elucidation of the mechanistic roles of Cx channels in lens cells will provide a better understanding of the key homeostatic process of ...