microinjection-recombinant-rcas-retrovirus-into-embryonic-chicken

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 ...

University of Texas Health Science Center, San Antonio

The present study was undertaken to explore the roles gap junctions play in lens epithelial cell differentiation.

Stimulation of lens cell differentiation by gap junction protein connexin 45.6.

Gap junctions and hemichannels are formed by a family of proteins called connexins. Till date up to twenty one different connexins have been characterized and their expression was observed to be spatio-temporally regulated. Gap junctions and hemichannels are involved in transfer of a variety of less than 1 kDa small molecules such as, ions, small metabolites, cAMP, ATP, IP3, prostaglandins, etc. Post-translational modifications of connexins and their interaction with other proteins are reported to be the key regulators of channel functions. Studies during the past decade or so, suggest the physiological important of connexin hemichannels mediating the communication between the cell and its environment. Molecules conveyed through the hemichannels elicit a variety of signaling pathways and influence cellular functions such as, cell cycle, tissue homeostasis, migration, mechanotransduction, oxidative stress. The purpose of the current review is to compile the reported studies so far and provide a general overview in our understanding how the molecular transfer through hemichannels regulates cellular signaling and functions.

Regulation of cellular function by connexin hemichannels.

Elevated oxidative stress (OS) during aging leads to bone loss. OS increases intracellular Ca(2+) ([Ca(2+)]i), resulting in cellular damage and death. We show earlier that Cx43 hemichannels open in response to OS, which serves as a protective mechanism for osteocytes. However, the underlying mechanism is unknown. Here, we found that treatment with H2O2 increased [Ca(2+)]i in osteocytes with [Ca(2+)]i being primarily derived from an extracellular Ca(2+) source. Hemichannel opening induced by OS was inhibited by the depletion of [Ca(2+)]i with BAPTA-AM, a Ca(2+) chelator, suggesting that [Ca(2+)]i influenced the activity of Cx43 hemichannels. Conversely, blockade of hemichannels had no effect on [Ca(2+)]i. A biotinylation assay showed that cell surface-expressed Cx43 was increased by OS, which could be inhibited by BAPTA-AM, suggesting that [Ca(2+)]i is necessary for Cx43 migration to the cell surface in response to OS. Together, these data suggest that increased hemichannel activity induced by OS was likely to be caused by elevated [Ca(2+)]i through increased Cx43 on the cell surface.

Elevated Intracellular Ca(2+) Signals by Oxidative Stress Activate Connexin 43 Hemichannels in Osteocytes.

Autophagy confers protective or detrimental effects on cells depending on the cellular context. We showed here that oxidative stress-induced cell death in osteocytic MLO-Y4 cells coincided with decreased autophagy. Decreased autophagy was also observed in osteocytes of superoxide dismutase 1- (SOD1-) deficient mice. Oxidative stress-induced osteocyte death was exacerbated by an autophagy inhibitor, chloroquine, suggesting a protective function of basal autophagy levels against oxidative stress-induced cell death. Pretreatment with dexamethasone reduced the susceptibility of osteocytes to oxidative stress-induced cell death and conferred protection against TNFα/cycloheximide-induced cell death. Inhibition of MAPK/ERK attenuated the formation of autophagosome, leading to increased osteocyte cell death. Taken together, our results suggest that autophagy, induced by moderate levels of glucocorticoids, leads to the preconditioning of osteocytes and conveys a novel cell-protective function against cell death induced by oxidative stress and other insults. © 2018 The Authors.  is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Jean X. Jiang

Department of Biochemistry and Structural Biology

University of Texas Health Science Center, San Antonio

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

Jean X. Jiang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Structural Biology

University of Texas Health Science Center, San Antonio

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

Department of Biochemistry and Structural Biology