Hypoxia-induced changes in Ca²⁺ mobilization and protein phosphorylation implicated in impaired wound healing

Abstract

Lee A, Derricks K, Minns M, Ji S, Chi C, Nugent MA, Trinkaus-Randall V. Hypoxia-induced changes in Ca 2ϩ mobilization and protein phosphorylation implicated in impaired wound healing. Am J Physiol Cell Physiol 306: C972-C985, 2014. First published March 26, 2014; doi:10.1152/ajpcell.00110.2013.-The process of wound healing must be tightly regulated to achieve successful restoration of injured tissue. Previously, we demonstrated that when corneal epithelium is injured, nucleotides and neuronal factors are released to the extracellular milieu, generating a Ca 2ϩ wave from the origin of the wound to neighboring cells. In the present study we sought to determine how the communication between epithelial cells in the presence or absence of neuronal wound media is affected by hypoxia. A signal-sorting algorithm was developed to determine the dynamics of Ca 2ϩ signaling between neuronal and epithelial cells. The cross talk between activated corneal epithelial cells in response to neuronal wound media demonstrated that injury-induced Ca 2ϩ dynamic patterns were altered in response to decreased O 2 levels. These alterations were associated with an overall decrease in ATP and changes in purinergic receptor-mediated Ca 2ϩ mobilization and localization of N-methyl-D-aspartate receptors. In addition, we used the cornea in an organ culture wound model to examine how hypoxia impedes reepithelialization after injury. There was a change in the recruitment of paxillin to the cell membrane and deposition of fibronectin along the basal lamina, both factors in cell migration. Our results provide evidence that complex Ca 2ϩ -mediated signaling occurs between sensory neurons and epithelial cells after injury and is critical to wound healing. Information revealed by these studies will contribute to an enhanced understanding of wound repair under compromised conditions and provide insight into ways to effectively stimulate proper epithelial repair. hypoxia; wound healing; imaging; cell communication DAMAGE TO TISSUE activates intricate underlying mechanisms that mediate the healing process. Communication between cells is generated immediately after injury and continues during the migration phase and the later phases of proliferation and extracellular matrix reassembly (13,14,23). This reepithelialization requires the precise control of glutamatergic and purinergic signaling pathways. Injured cells release nucleotides that serve as ligands for cell-surface purinergic receptors, and injured neurons release components, including ATP and glutamate, that bind their respective receptors. The release of nucleotides can modulate Ca 2ϩ homeostasis (4,11,43,52,54).Neuronal transmitters such as glutamate bind to metabotropic G protein-coupled receptors or to ionotropic receptors, including 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptors. It has been reported that epithelial cells do not respond to AMPA or kainate, but they do respond to glutamate in neuronal wound media and to NM...

Purinoreceptor P2X7 Regulation of Ca2+ Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury

“…6,16 Briefly, Sprague-Dawley juvenile rats (Charles River Labs, Wilmington, MA) were euthanized, and heads were kept on ice until corneal dissection. A 3-mm-diameter region in the center of the cornea was demarcated with a trephine, and the epithelium was removed from the region.…”

“…Normalized concentrations were reverse transcribed, as previously described. 6 Quantitative real-time PCR was performed using verified TaqMan probes (Life Technologies, Grand Island, NY) for target or 18S on an ABI 7300 thermal cycler (Applied Biosystems, Foster City, CA). Relative expression was determined using the DDCt method, using 18S to normalize.…”

“…Immunofluorescence staining was performed as described previously. 5,6 Briefly, cells or tissues were permeabilized with 0.1% v/v Triton X-100 in PBS for 2 to 5 minutes and blocked with 4% bovine serum albumin in PBS for 1 hour. Cells or tissues were incubated in primary antibody solutions overnight at 4 C. Alexa Fluoreconjugated secondary antibodies (Invitrogen) were used at a dilution of 1:100 in blocking solution for 1 hour at room temperature.…”

“…6,18 Briefly, cells were cultured to confluence onto fetal bovine serumecoated glass coverslips (for perfusion) or glass bottom dishes (for wounding). Growth supplements were removed 16 to 24 hours before experimentation.…”

“…3 Although downregulation of specific P2 receptors decreases Ca 2þ mobilization in corneal epithelial cells, 4 activation of P2 purinergic receptors by nucleotides causes increased phosphorylation of adaptor proteins and focal adhesion kinases. 5,6 These changes mediate signaling events that regulate migration, demonstrating the critical role of nucleotide signaling in wound repair.…”