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 signaling pathways. To identify potential regulators of UVB-induced apoptosis, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry fingerprint analysis to identify proteins that are altered in the rafts after UVB irradiation. Our data shows 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.

Background

Lipid rafts are - sphingolipid- and cholesterol-enriched microdomains of the cellular membrane that are characterized by light buoyant density using density gradient centrifugation while treated with non-ionic detergent Triton X-100 at the low temperature [1, 2]. Several apoptotic regulatory proteins, such as Fas, Ras, and TNFα, have been shown to be recruited into lipid rafts upon various stimuli [3, 4]. Ultraviolet light (UV) is one of the stimuli that alters major components of lipid rafts, which induces expression of various genes and regulates apoptosis through recruiting and promoting the aggregation of Fas, formation of death-inducing signaling complex (DISC) and activation of Fas-FADD (Fas-associated death domain protein)-caspase 8 cascade in skin cells [5-8]. However, the functions of other lipid rafts-associated proteins in regulation of UVB-induced apoptosis are still generally unknown. In this report, proteomic screening was used to identify lipid rafts-associated proteins in human keratinocytes HaCaT cells in the presence and absence of UVB-irradiation. A potential apoptosis regulator, prohibitin (PHB), was increased in lipid rafts after UVB irradiation. Further analysis indicated that PHB, a lipid rafts-associated anti-apoptotic protein [9-16], plays a role in regulation of UVB-induced apoptosis of keratinocytes.

Questions Addressed

Lipid rafts-association of prohibitin and its role in protecting HaCaT cells from apoptosis upon UVB-irradiation.

Experimental Design

Western blot analysis

Antibodies against PHB (sc-28259) and caveolin-1 (sc-894) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-β-actin (A5316) was purchased from Sigma-Aldrich (St. Louis, MO). The proteins were separated on SDS-PAGE, transferred to a nitrocellulose membrane and probed with proper first and secondary antibodies. The signal was developed using West Pico Supersignal Chemilluminescent kit (Pierce, Rockford, IL).

PHB silencing using RNA interference

Cells were seeded in antibiotic-free DMEM with 10% fetal bovine serum (FBS) and incubated at 37 °C with 5% CO₂ until 60-80% confluent. The medium was then replaced with a transfection mixture (500 μL), containing scrambled siRNA (60 nM, sc-37007) or PHB siRNA (60 nM, sc-37629), siRNA Transfection Medium and siRNA Transfection Reagent, prepared according to manufacture's protocol (Santa Cruz Biotechnology, Santa Cruz, CA). The cells were then cultured at 37 °C with 5% CO₂ for 6 h before antibiotic-free DMEM with 20% FBS (500 μL) was added to each well for overnight incubation. The transfection reagent was then replaced with DMEM with 10% FBS and incubated for another 18-24 h before harvesting.

Apoptosis assay

The apoptotic cell death was determined using a fluorescein isothiocyanate (FITC)-conjugated annexin V (ANX5)/propidium iodide (PI) apoptosis detection kit following the manufacturer's protocol (BD Biosciences, Franklin Lakes, NJ). Briefly, the cells were suspended in annexin V binding buffer at 1×10⁶ cells/mL. The cell suspension (100 μL) was mixed with 5 μL FITC-ANX5 and 5 μL PI. After incubating in the dark for 15 min, the cells were combined with 400 μL ANX5 binding buffer and apoptotic death was analyzed using a FACSort Flow Cytometer (BD Biosciences, Franklin Lakes, NJ) equipped with CellQuest software (BD Biosciences, Franklin Lakes, NJ). 1×10⁵ cells were collected and analyzed.

Results

Translocation of PHB after UVB irradiation

2-DE analysis revealed that PHB is enriched in lipid rafts after UVB irradiation (Fig. S1). To further analyzing the effect of UVB on PHB distribution, western blot was used to determine PHB and caveolin-1 in each fraction of the density gradient. Our data showed that caveolin-1, a protein marker present in lipid rafts [17], was predominantly located in Fraction 2 of the density gradient (Fig. 1A). The level of caveolins-1 in Fraction 2 and the levels of total protein in each fraction were not significantly changed after UVB irradiation (Figs. 1A, S1), which agreed with our previous observation [7]. The semi-quantitative analysis of the blot revealed that the percentage of PHB in lipid raft fraction was increased from 45% to 74% after UVB irradiation (Fig. 1B). The result indicates that UVB irradiation induces PHB translocation from non-raft fractions into the lipid raft fraction.

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.

The role of PHB in regulation of UVB-induced apoptosis

PHB can protect cells from oxidative stress-induced cell injury and apoptosis [13-15, 18, 19]. Since oxidative stress plays a critical role in UVB-induced cell death [20, 21], we determined whether PHB also played a role in regulation of UVB-induced apoptosis. PHB siRNA was used to reduce the expression of PHB in HaCaT cells. Our data show that while the expression of PHB was not affected by scrambled siRNA, it was significantly reduced by PHB siRNA in the presence or absence of UVB irradiation (Fig. 2A). The reduced expression of PHB did not have a statistically significant effect on cell apoptosis (8.9±2.5%) without UVB irradiation (Fig. 2B). However, the cells transfected with PHB siRNA were more sensitive to UVB-induced apoptosis (48.6±7.2%) compared to the cells that were transfected with scrambled siRNA (34.2±1.0%) (Fig. 2B). These results suggest that PHB plays a protective role in UVB-induced skin cell apoptosis.

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.

Conclusion

PHB, a mitochondria-associated protein, plays a critical role in regulation of many cell functions, such as cell proliferation [22], cell apoptosis [23], cell-cycle control [24], cell senescence, development, and tumor suppression [25]. Previous research has shown that PHB is important in both cell-cycle control and mitochondrial integrity control in keratinocytes [26]. PHB is also essential in cell signaling pathways outside of the mitochondria, such as Raf-ERK [27] and PI3K/Akt signaling cascades [28]. The potential involvement of PHB in UVB-induced apoptosis is reported here for the first time. Our data showed that the cellular distribution of PHB was significantly altered after UVB irradiation (Fig. 1). While the expression of PHB was not significantly changed (Fig. 2A, Lane 4 vs. 1), the lipid rafts-associated PHB was increased after UVB irradiation (Fig. 1A, Fraction 2, UVB vs. Control). Reducing the expression of PHB using PHB siRNA increased the number of apoptotic cells after UVB irradiation, but had little effect on cells without the irradiation (Fig. 2B). These results indicate that PHB is translocated to lipid rafts and protects cells from apoptosis upon UVB irradiation. However, the role of lipid rafts translocation of PHB in UVB-induced apoptosis remains undetermined.

ABBREVIATIONS

UVB

ultraviolet B light (290–320 nm)

PHB

Prohibitin