Expression and functional significance of phosphoenolpyruvate carboxykinase 1 in uveal melanoma.

Uveal melanoma (UVM), an uncommon yet potentially life-threatening ocular cancer, arises from melanocytes in the uveal tract of the eye. The exploration of novel oncotargets for UVM is of paramount importance. In this study, we show that PCK1 (phosphoenolpyruvate carboxykinase 1) expression is upregulated in various UVM tissues as well as in primary UVM cells and immortalized lines. Furthermore, bioinformatics studies reveal that PCK1 overexpression in UVM correlates with advanced disease stages and poor patient survival. Genetic silencing (utilizing viral shRNA) or knockout (via CRISPR/Cas9) of PCK1 significantly curtailed cell viability, proliferation, cell cycle progression, and motility, while provoking apoptosis in primary and immortalized UVM cells. Conversely, ectopic overexpression of PCK1, achieved through a viral construct, bolstered UVM cell proliferation and migration. Gαi3 expression and Akt phosphorylation were reduced following PCK1 silencing or knockout, but increased after PCK1 overexpression in UVM cells. Restoring Akt phosphorylation through a constitutively active mutant Akt1 (S473D) ameliorated the growth inhibition, migration suppression, and apoptosis induced by PCK1 silencing in UVM cells. Additionally, ectopic expression of Gαi3 restored Akt activation and counteracted the anti-UVM cell effects by PCK1 silencing. In vivo, the growth of subcutaneous xenografts of primary human UVM cells was significantly inhibited following intratumoral injection of adeno-associated virus (aav) expressing PCK1 shRNA. PCK1 depletion, Gαi3 downregulation, Akt inhibition, proliferation arrest, and apoptosis were detected in PCK1-silenced UVM xenografts. Collectively, our findings demonstrate that PCK1 promotes UVM cell growth possibly by modulating the Gαi3-Akt signaling pathway.

Alpha-melanocyte stimulating hormone protects retinal pigment epithelium cells from oxidative stress through activation of melanocortin 1 receptor-Akt-mTOR signaling.

Patients with age related macular degeneration (AMD) will develop vision loss in the center of the visual field. Reactive oxygen species (ROS)-mediated retinal pigment epithelium (RPE) cell apoptosis is an important contributor of AMD. In this study, we explored the pro-survival effect of α-melanocyte stimulating hormone (α-MSH) on oxidative stressed RPE cells. We found that α-MSH receptor melanocortin 1 receptor (MC1R) was functionally expressed in primary and transformed RPE cells. RPE cells were response to α-MSH stimulation. α-MSH activated Akt/mammalian target of rapamycin (mTOR) and Erk1/2 signalings in RPE cells, which were inhibited by MC1R siRNA knockdown. α-MSH protected RPE cells from hydrogen peroxide (H2O2)-induced apoptosis, an effect that was almost abolished when MC1R was depleted by siRNA. α-MSH-mediated S6K1 activation and pro-survival effect against H2O2 was inhibited by Akt inhibitors (perifosine, MK-2206 and LY294002). Further, mTOR inhibition by rapamycin, or by mTOR siRNA knockdown, diminished α-MSH's pro-survival effect in RPE cells. Thus, Akt and its downstream mTOR signaling mediates α-MSH-induced survival in RPE cells. In summary, we have identified a new α-MSH-MC1R physiologic pathway that reduces H2O2-induced RPE cell damage, and might minimize the risk of developing AMD.

Ultraviolet (UV) and hydrogen peroxide activate ceramide-ER stress-AMPK signaling axis to promote retinal pigment epithelium (RPE) cell apoptosis.

Ultraviolet (UV) radiation and reactive oxygen species (ROS) impair the physiological functions of retinal pigment epithelium (RPE) cells by inducing cell apoptosis, which is the main cause of age-related macular degeneration (AMD). The mechanism by which UV/ROS induces RPE cell death is not fully addressed. Here, we observed the activation of a ceramide-endoplasmic reticulum (ER) stress-AMP activated protein kinase (AMPK) signaling axis in UV and hydrogen peroxide (H2O2)-treated RPE cells. UV and H2O2 induced an early ceramide production, profound ER stress and AMPK activation. Pharmacological inhibitors against ER stress (salubrinal), ceramide production (fumonisin B1) and AMPK activation (compound C) suppressed UV- and H2O2-induced RPE cell apoptosis. Conversely, cell permeable short-chain C6 ceramide and AMPK activator AICAR (5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide) mimicked UV and H2O2's effects and promoted RPE cell apoptosis. Together, these results suggest that UV/H2O2 activates the ceramide-ER stress-AMPK signaling axis to promote RPE cell apoptosis.

Protective role of Wallerian degeneration slow (Wld(s)) gene against retinal ganglion cell body damage in a Wallerian degeneration model.

Nerve distal axon injury-induced Wallerian degeneration is significantly delayed in Wallerian degeneration slow (Wld(s)) mutant mice, although the associated mechanisms are not completely clear and the role of Wld(s) in retinal ganglion cell (RGC) body damage is not fully understood. In the present study, a Wallerian degeneration model was established in wild-type (WT) and Wld(s) mutant mice by creating mechanical injury in the optic nerves. Wallerian degeneration and RGC body collapse were observed to be significantly delayed in the Wld(s) mice. Electroretinograms (ERG) and visual evoked potentials (VEPs) in Wld(s) mice were also significantly improved at the earlier stages (one week) following injury. The retina immunohistochemistry results showed that Wld(s) mice had more ordered cells and improved inner granular cell layer arrangement compared with the WT mice. Optic nerve Luxol Fast Blue (LFB) staining showed greater axon demyelination in WT mice than in Wld(s) mice. A large number of apoptotic cells were also observed in the WT mice. The present results suggest that the Wld(s) gene may also protect the RGC body following nerve injury.