Cigarette smoke affects keratinocytes SRB1 expression and localization via H2O2 production and HNE protein adducts formation

Abstract

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H(2)O(2)) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H(2)O(2), induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake.

Figure 1. SR-B1 is expressed in epidermis of mouse (A) and human (B) skin.

The anti SR-B1 antibody dekorates/stains most nuclei of keratinocytes (as the ones indicated by the arrows) in both murine and human normal skin SR-B1 immunohistochemistry, DAB, Original Magnification ×200 (human skin) and ×400 (mouse skin).

Figure 2. Exposure to CS decreased SR-B1 protein levels in HaCaT cells. Cells were exposed to CS for 50 min and cells were harvested at different time points (0–24 hrs).

The Western blot shown in the top is representative of five experiments. Quantification of the SR-B1 bands is shown in the bottom panel. Data are expressed in arbitrary units (averages of five different experiments, *p<0.05). β-actin was used as loading control. Immunogold for SR-B1 confirm the decreased protein levels after CS exposure (B). IHC for SR-B1 is shown in the C panel (arrows).

Figure 3. Cigarette Smoke exposure induces changes in SR-B1 levels and localization in human keratinocytes.

Immunocytochemistry of HaCaT cells showing localization of SR-B1 (green) before and after CS exposure for different time points. Images are merged and are representative of at least 100 cells viewed in each experiments (n = 5). Nuclei (blue) were stained with DAPI. A) Cells overview at different time points 40×; B) Representative Western blot of proteins extracted from the membranes of cells exposed to Cigarette Smoke at different time points. The signals of SR-B1 protein levels were determined by densitometric analysis of the scanned images (bottom panel). Data are expressed in arbitrary units and are averages of the values for five different experiments. (*p<0.05).

Figure 4. Exposure to CS increased HNE (A) and ACR protein adducts (B) in HaCaT cells measured by Western blot and this is confirmed also by ICC staining for HNE and ACR (C) (see arrows).

CS increased carbonyl groups expression (D) in HaCaT cells. Cells were exposed to CS for 50 min and cells were harvested at different time points (0–12 hrs). Western blot shown in the top is representative of five experiments. Quantification of the SR-B1 bands is shown as ratio of SRB1/β-actin (bottom panel). Data are expressed as arbitrary units (averages of five different experiments, *p<0.05; **p<0.01). β-actin was used as loading control.

Figure 5. HNE (A) or ACR (B) treatment did not affect SR-B1 levels in HaCaT cells.

Cells were exposed to the different treatments for 50 min and cells were harvested at different time points (0–24 hrs). Numbers below the blot represent the ratio of SRB1/β-actin quantification.

Figure 6. CS induces the increase of HNE/SRB1 adducts.

Immunocytochemistry of HaCaT cells showing localization of HNE-adducts (left column, green color), SR-B1 (central column, red color) and HNE/SR-B1 adducts (right column, yellow color) before, and several time points after, CS exposure (A). Images are merged in the right panel and the yellow color indicates overlap of the staining. These data were confirmed by immunoprecipitation for SR-B1 (B). HaCaT cells were exposed to CS and cell lysates were immunoprecipitated using anti SR-B1. Immunoprecipitated proteins were separated by SDS-PAGE, and then transferred to a nitrocellulose membrane and immunoblotted with anti-HNE. Western blot shown is representative of five independent experiments.

Figure 7. CS induces the increase of Ubiquitin/SR-B1 adducts.

HaCaT cells were exposed to CS and cell lysates were immunoprecipitated using anti SR-B1. Immunoprecipitated proteins were separated by SDS-PAGE, and then transferred to a nitrocellulose membrane and immunoblotted with anti-Ubiquitin (A). Pretratment (2 h) with MG-132 (proteosome inhibitor) did not affect SR-B1 levels. Cells were exposed to CS for 50 min and harvested at different time points (0–24 hrs). Western blot shown in the top is representative of five independent experiments. Quantification of the SR-B1 bands is shown as ratio of SR-B1/β-actin (bottom panel). Data are expressed as arbitrary units (averages of five different experiments). β-actin was used as loading control.

Figure 8. GO treatment decreased SR-B1 levels.

Cells were treated with GO for 50 min and then harvested at different time points (0–24 hrs). A) Representative Western blot of five independent experiments is shown in the top panel. Quantification of the SRB1 bands, average of the five independent experiments, is shown in the bottom panel. Data are expressed in arbitrary units (**p<0.01). β-actin was used as loading control. B) Concentration of H₂O₂ level in cell treated with GO. Data are presented as average of triplicate measurements from each sample and expressed as arbitrary units.

Figure 9. CS exposure increased H2O2 levels and mitochondrial superoxide production.

Cells were exposed to CS for 15, 30 or 50 min. (A) Concentration of H₂O₂ in the media with (close bars) or without cells (open bars). Data are presented as average of triplicate measurements from each sample and expressed as arbitrary units. (B) Mitochondrial ROS production was evaluated by Mitosox fluorescence. Cells were loaded with Mitosox before and after CS exposure and subjected to live cell imaging.

Figure 10. Exposure to CS increased NADPH oxidase levels in HaCaT cells. Cells were exposed to CS for 50 min and cells were harvested at different time points (0–24 hrs).

The activation of NADPH oxidase was determined by the translocation in membrane of p67^phox (A) and p47^phox (B). The Western blot shown in the top is representative of five experiments. Quantification of the SR-B1 bands is shown in the bottom panel. Data are expressed as arbitrary units (averages of five different experiments, *p<0.05). β-actin was used as loading control. These data were confirmed by ICC for p6^phox and p4^phox (C).

Figure 11. The decreased levels of SR-B1 after CS exposure was reversed by catalase (CAT) (left panel) or Diphenyleneiodonium Chloride (DPI) (right panel).

Cells pretreated with CAT or DPI were exposed to CS for 50 min and harvested at different time points (0–24 hrs). Western blot shown is a representative of five independent experiments. Quantification of the SR-B1 bands is expressed under the blot as ratio of SR-B1/β-actin (arbitrary units).

Figure 12. Possible mechanism involved in the degradation of SR-B1.

Among the components present in CS there are acrolein and H₂O₂ that beside to react with the membrane lipids (1) are able to cross the cell membrane (2), once H₂O₂ is inside the cells, there will be the formation of OH. (Fenton reaction) (3) that will react with the cytosolic membrane lipids and the formation of lipid peroxidation products such as ACR and HNE (4). ACR and HNE can from SR-B1 adducts (5 and 6) and HNE can also activate NOX by inducing the translocation of the cytoplasmic submit to the membrane (7). Activation of NOX lead to the increased production of O₂ ⁻ that can be dismutated (SOD) in H₂O₂ (8) that via Fenton reaction will further increase the level of peroxidation (9). The formation of HNE-SR-B1 adducts is recognized by the ubiquitination apparatus of the cell (10) that will ubiquitinate the protein that subsequent will be dregraded by the proteosome (11).