Lipid rafts association and anti-apoptotic function of prohibitin in ultraviolet B light-irradiated HaCaT keratinocytes

Abstract

Upon UVB irradiation, an alternation of major lipid raft components can lead to the recruitment/activation of rafts-associated proteins and initiation of downstream apoptotic signalling pathways. We used two-dimensional gel electrophoresis (2-DE) to identify potential regulators of UVB-induced apoptosis and mass spectrometry fingerprint analysis to identify proteins that are altered in the rafts after UVB irradiation. Our data show that levels of several proteins, including prohibitin (PHB), were changed in lipid rafts after UVB irradiation. We also demonstrate that while total PHB expression was not changed, the protein was enriched in lipid rafts after UVB irradiation. Reduced expression of PHB using siRNA knockdown resulted in an increase in cellular apoptosis after UVB irradiation. Based on these results, we propose that PHB protects keratinocytes from UVB-induced apoptosis.

Figure 1

Redistribution of PHB in HaCaT cells after UVB irradiation. HaCaT cells were treated or not treated with UVB (50 mJ/cm²). At 6 h post-UVB, the cells were lysed and lipid rafts from equal amount of proteins were isolated using Optiprep™ density gradient ultracentrifugation (1.2 mL 5%-1.6 mL 50%-1.2 mL 40%). Panel A: western blot analysis of PHB in each fraction. 30 μL of fractions (1, 3-5), 15 μL of fractions (2) and 3 μL of total lysates were subjected to the analysis. Panel B, the intensity of PHB band in each fraction was analyzed using ImageJ (Version 1.45, NIH). The data in this panel represents the average of three independent experiments.

Figure 2

PHB protects HaCaT cells from UVB-induced apoptosis. HaCaT cells were transiently transfected with scrambled siRNA or PHB siRNA. The cells without transfection were used as control. At 40 h post-transfection, the cells were UVB-irradiated (50 mJ/cm²). The cell apoptosis was analyzed at 6 h post-UVB using AXVFITC/PI double staining followed flow cytometry analysis. The percentage of apoptotic cells was calculated using CellQuest software by dividing the total apoptotic cells (double stained cells) with total number of counted cells. The data represents the means ± SD of 3 independent experiments. Student's t-test was used to analyze the data and p value less than 0.05 was considered as statistically significant. *: p<0.05 PHB siRNA vs. Scrambled siRNA.