Hyperosmotic Stress Reduces Melanin Production by Altering Melanosome Formation

<div><p>Many tissues of the human body encounter hyperosmotic stress. The effect of extracellular osmotic changes on melanin production has not yet been elucidated. In this study, we determined that hyperosmotic stress induced by organic osmolytes results in reduced melanin production in human melanoma MNT-1 cells. Under hyperosmotic stress, few pigmented mature melanosomes were detected, but there was an increase in swollen vacuoles. These vacuoles were stained with an anti-M6PR antibody that recognizes late endosomal components and with anti-TA99 and anti-HMB45 antibodies, implying that melanosome formation was affected by hyperosmotic stress. Electron microscopic analysis revealed that the M6PR-positive swollen vacuoles were multi-layered and contained melanized granules, and they produced melanin when L-DOPA was applied, indicating that these vacuoles were still capable of producing melanin, but the inner conditions were not compatible with melanin production. The vacuolation phenomenon induced by hyperosmotic conditions disappeared with treatment with the PI3K activator 740 Y-P, indicating that the PI3K pathway is affected by hyperosmotic conditions and is responsible for the proper formation and maturation of melanosomes. The microarray analysis showed alterations of the vesicle organization and transport under hyperosmotic stress. Our findings suggest that melanogenesis could be regulated by physiological conditions, such as osmotic pressure.</p></div

PI3K activation inhibits hyperosmotic stress-induced vacuolation.

<p>MNT-1 cells were treated with 50 mM sucrose, 10 µM wortmannin, 10 µM YM201636, or 20 µM 740 Y-P for 24 hours. After fixation in 3% paraformaldehyde and permeabilization with 0.1% Triton X-100 in phosphate-buffered saline (PBS), the cells were stained with an antibody against M6PR and examined using confocal microscopy at a magnification of 1260×. NC, negative control; DIC, differential interference contrast.</p

Hyperosmotic stress down-regulates melanin production.

<p>Hyperosmotic stress reduces melanin production but does not affect tyrosinase activity in MNT-1 cells. (A) MNT-1 cells were treated with 50 mM of the indicated sugar for 7 days, and the color of the cell pellets was monitored. NC, negative control. (B) The melanin content was measured at 450 nm. The data are representative of three independent experiments (***, P < 0.005). (C) Tyrosinase activity assay. MNT-1 cell extracts were incubated with 10 mM L-dihydroxyphenylalanine (L-DOPA), and the amount of melanin was measured at 450 nm. The data are representative of three independent experiments (N.S., not significant).</p

Hyperosmotic stress-induced M6PR-positive swollen vacuoles contain TYRP-1 and PMEL17.

<p>MNT-1 cells were treated with 50 mM sucrose for 7 days and stained with anti-TA99 (A) or anti-HMB45 (B) antibodies. The fluorescence images were acquired using confocal microscopy at a magnification of 1260×. The insets show the magnified images. NC, negative control. (C) Electron microscopic analyses were performed on MNT-1 cells before and after 50 mM sucrose treatment for 7 days. For the in situ L-DOPA assay, the MNT-1 cells were incubated with 0.1% L-DOPA for 3 hours after treatment with 50 mM sucrose for 7 days (Sucrose + DOPA). The insets show the magnified images. The arrowheads indicate various stages of melanosomes (NC), swollen vesicles containing dark granules (Sucrose), or swollen vesicles after the in situ DOPA assay (Sucrose + DOPA). The dotted lines identify the position for the magnified images. NC, negative control.</p

Hyperosmotic stress induces vacuolation.

<p>Hyperosmotic stress induces swelling of M6PR-positive vacuoles. (A) MNT-1 cells were treated with 50 mM sucrose for 12 hours. After fixation in 3% paraformaldehyde and permeabilization with 0.1% Triton X-100 in phosphate-buffered saline (PBS), the cells were stained with an anti-M6PR antibody and examined by confocal microscopy at a magnification of 400×. NC, negative control; DIC, differential interference contrast. (B) MNT-1 cells were treated with 50 mM sucrose for 7 days and stained with the intracellular vesicle markers anti-EEA1, anti-M6PR or anti-LAMP. The images were acquired using a confocal microscope at 1260×.</p

Schematic model of hypo-pigmentation under hyperosmotic stress.

<p>Hyperosmotic stress induces swollen, abnormal melanosomes in which the inner condition is not appropriate for melanin production. The PI3K pathway is involved in proper vesicle trafficking for melanosome formation and maturation. Thus, disruption of this pathway by sucrose-induced hyperosmotic stress results in the formation of swollen, abnormal melanosomes.</p

Genome-wide analysis reveals that hyperosmotic stress affects the expression of genes involved in intracellular vesicle transport.

<p>(A) log₂-fold-changes of up-regulated (red) or down-regulated (green) genes were displayed after hyperosmotic stress by sucrose treatment. The cells were treated with 50 mM sucrose for 7 days, and the microarray analysis was performed using 10 µg of the total RNA. The color bar represents gradients of log2-fold-changes in each comparison. NC, negative control. (B) The biological processes that were significantly up-regulated in sucrose-treated cells were summarized after gene ontology (GO) analysis.</p

Discovery, Optimization, and Biological Evaluation of Sulfonamidoacetamides as an Inducer of Axon Regeneration

Axon regeneration after injury in the central nervous system is hampered in part because if an age-dependent decline in the intrinsic axon growth potential, and one of the strategies to stimulate axon growth in injured neurons involves pharmacological manipulation of implicated signaling pathways. Here we report phenotypic cell-based screen of chemical libraries and structure–activity-guided optimization that resulted in the identification of compound <b>7p</b> which promotes neurite outgrowth of cultured primary neurons derived from the hippocampus, cerebral cortex, and retina. In an animal model of optic nerve injury, compound <b>7p</b> was shown to induce growth of GAP-43 positive axons, indicating that the in vitro neurite outgrowth activity of compound <b>7p</b> translates into stimulation of axon regeneration in vivo. Further optimization of compound <b>7p</b> and elucidation of the mechanisms by which it elicits axon regeneration in vivo will provide a rational basis for future efforts to enhance treatment strategies