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The epidermis is the outermost protection of the organism. As so, defence program has to be initiated in stress situation in order to protect keratinocytes. The EGF receptor (EGFR) controls cell proliferation and migration in keratinocytes, being a major regulator of keratinocyte homeostasis within the epidermis. The EGFR is known to be internalized without addition of ligand under the control of p38 MAPK during stress conditions in HeLa cells, but also following lipid rafts disruption in keratinocytes. This could represent an alternative internalization process that removes the EGFR from cell surface. Here, we investigated whether other stress conditions such as scratch wounding keratinocyte monolayer or incubation with a sensitizer chemical (i.e. DNFB), could also induce this peculiar mechanism of EGFR internalization. Our results show that both stressing conditions induce p38 MAPK activation concomitantly with EGFR internalization, independently of ligand binding to the EGFR. Inhibition of p38 MAPK activity during scratch wound blocks EGFR internalization at the margin of the wound while cell migration is impeded. Our results show thus that the p38 MAPK-dependent EGFR internalization is a process shared by keratinocytes when submitted to challenging conditions.

The receptor for epidermal growth factor (EGF) plays an important role in epidermal keratinocytes and is known to move out of lipid raft after cholesterol depletion, leading to ligand-independent activation. Accumulation of evidence indicates the ability of EGF receptor (EGFR) to undergo internalization without participation of the ligand under the control of p38 MAPK during stress conditions. Since cholesterol depletion using methyl-beta-cyclodextrin is known to induce ligand-independent activation of EGFR in keratinocytes, we investigated by confocal microscopy and ligand-binding tests the processing and localization of EGFR following lipid raft disruption. Here, we report the dimerization and the slow internalization of the receptor accompanied by the delayed phosphorylation of tyrosine 1068 and its degradation by the proteasome. We also demonstrate the involvement of p38 MAPK during the process of internalization, which can be considered as a protective response to stress. Moreover, cholesterol-depleted keratinocytes recover their ability to proliferate during the recovery period that follows lipid raft disruption.

Following injury induced by oxidant stress (exposure to xanthine-xanthine oxidase during 120 min), cultured human umbilical vein endothelial cells were severely modified. These lesions were evaluated by electron microscopy and the types of injury were shown to be progressive cell blebbing as well as altered mitochondrial features. Enlargement of the endoplasmic reticulum and swelling of the Golgi apparatus were also observed. With the intention of learning more about the role of oxygen-derivative-induced alterations of the endocytotic apparatus (plasma membrane, endosomes, lysosomes), the receptor-mediated endocytosis of colloidal gold-conjugated LDL was evaluated. It was demonstrated that this endocytosis is drastically decreased following this type of injury, corroborating our previous report of important reduction in 125I-LDL endocytosis under the same conditions. Tubular electron-lucent structures, often observed in the vicinity of blebs, could possibly be involved in the impairment of LDL receptor accessibility.

We used electron microscopy serial sections and acid phosphatase cytochemistry to follow the endocytosis of low density lipoproteins (LDL) by cultured human umbilical vein endothelial cells. The surface LDL-receptors were labeled with LDL-colloidal gold conjugates at 4 degrees C and then, cells were incubated for 0, 5, 10, 20, 30 and 60 min at 37 degrees C before fixation. The organelles identified in this way and studied by serial sections corresponded to those described in endothelial cells and in other cell types, with a peripheral endosomal compartment and a juxtanuclear endosomal compartment, located in the vicinity of the Golgi apparatus and mainly composed of round vesicle-containing structures called multivesicular bodies. By acid phosphatase cytochemistry, the limits between the endosomal juxtanuclear compartment and the lysosomal compartment were also studied and it was shown that multivesicular bodies are probably the first site for the acquisition of acid hydrolases. Lastly, the observation of highly gold-labeled endothelial cells when cells were incubated for 90 min at 37 degrees C with the LDL-gold conjugate, suggested that LDL was degraded in the lysosomal compartment.

Endothelial cells (EC) of blood vessels are submitted to oxidative stress under various circumstances. These conditions may modify EC functions; therefore, in the present work we have studied the receptor-mediated endocytosis of low-density lipoproteins (LDL) and malondialdehyde-modified LDL by the LDL receptor and the "scavenger" receptor, respectively, in cultured human umbilical vein EC after short (0-120 minutes) incubations in a superoxide anion (O2-) generating system. In both receptor-mediated processes, the oxidative stress produces a significant decrease at four different LDL concentrations (5-50 micrograms/ml) after 120 minutes of oxidation. On the other hand, the fluid-phase endocytosis of sucrose by EC seems to be stimulated by these conditions. Furthermore, incorporation of antioxidant enzymes in the O2- -producing system shows that H2O2 is an obligatory intermediate in order to produce the effect on the receptor-mediated processes. Hypotheses concerning the mechanisms involved in the modifications of endocytotic processes and their implications in vivo are discussed.