| Background:Ultraviolet (UV), which is an important environmental factors, is closely related with human skin. According to the wavelength, UV can be divided into ultraviolet A (UVA, 320-400nm), ultraviolet B (UVB,280-320nm) and ultraviolet C (UVC,200-280nm). Among them, only UVA and UVB could reach the Earth's surface. Compared to the same dose of UVA, UVB is 1000 times powerful and can induce some obvious damages to skin.Fibroblasts are major component cells in dermis. After UVA or UVB irradiation, their growth, differentiation and function will change significantly. In previous study, we found that small dose of UVB radiation increased vimentin protein content, which is a specific cytoskeleton in dermal fibroblast. There are some enzyme cutting sites in vimentin, which could be recognized by caspase-8 and-3. During cell apoptosis, different caspases might disrupt vimentin and its cleaved products promote apoptosis.Objective:(1) To explore whether caspase-8 or-3 could be activated and the changes of receptor interacting protein-1 (RIP-1) in UVB induced apoptosis of skin fibroblasts.(2) To investigate fibroblast-specific vimentin could be cleaved by different caspases.(3) To compare the changes of the other two cytosketal proteins tubulin and actin in theapoptotic fibroblasts.(4) To elucidate the mechanism of apoptosis induced by UVB in skin fibroblasts.Methods:(1) Primary human skin fibroblasts were cultured in vitro and subcultured, the 3 to 6 passages of cells were used in experiments. (2) Cell viability affected by different doses of UVB was measured by the 3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-diphenytetrazoliumromide (MTT) assay. At 12h,24h,36h and 48h after 150mJ/cm2 UVB irradiation, cell viability was also detected by MTT assay. (3) Through Hoechst staining, the morphological changes of nuclear in cells irradiated by 150mJ/cm2 UVB were observed and the numbers of apoptotic cells were counted. (4) By caspase activity assays, the activity changes of caspase-8 or-3 in 150mJ/cm2 UVB-induced apoptotic cells. (5) Immunofluorescence and Western Blot were used to detect the protein levels of vimentin. (6) Western Blot was performed to compare the changes of a-tubulin,β-tubulin andβ-actin protein content in cells irradiated by 150mJ/cm2 UVB. (7) RT-PCR and Western Blot were used to determine the mRNA and protein levels of RIP-1.Results:(1) Cell viability of human skin fibroblasts was lowered to different extents at 24h after 50-300mJ/cm2 UVB radiation. When cells were treated with 150mJ/cm2UVB, cell viability decreased at 12h,24h,36h and 48h after treatment.(2) 150mJ/cm2UVB could induce cell apoptosis, and when the activity of caspases was inhibited, UVB-related apoptotic cell numbers decreased.(3) Caspase-8 and-3 were activated in fibroblasts irradiated by UVB, and their activities changed in a time dependent way. At 12h afterirradiation, their activities were obviously higher than that of other time points.(4) When caspase-8 activity was inhibited, the activity of caspase-3 was lowered; similarly, when caspase-3 activity was inhibited, the process of caspase-8 activation was delayed.(5) When the activity of caspase-8 or-3 was inhibited, the proportion of apoptotic fibroblasts reduced.(6) In UVB-related apoptotic skin fibroblasts, vimentin showed an enhanced expression,β-tubulin showed a lowered expression, and both of the contents of a-tubulin and P-actin remained unchanged.(7) In the process of UVB-induced apoptosis, RIP-1 mRNA was down-regulated and its protein levels were up-regulated.Conclusions:(1) Activated caspase-8 and-3, which influenced each other, played important roles in UVB-triggered apoptotic fibroblasts.(2) UVB-activated caspase-8 and-3 did not disrupt their substrate vimentin to promote fibroblasts apoptosis.(3) Decreasedβ-tubulin and increased RIP-1 may play some roles in apoptosis of skin fibroblasts(4) In UVB-irradiated fibroblasts, the protein levels ofα-tubulin andβ-actin did not affected by UVB, and both of them could be used as internal controls in immunoblotting.
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