The effects of low-doses UVA irradiation on HaCaT keratinocytes

Description

We examined the effects of low-doses UVA irradiation on HaCaT keratinocytes with the aim of identifying UVA-induced effects on cell motility proteins. For this purpose, a comparative proteomic analysis of HaCaT keratinocytes was performed before and after irradiation with two cumulative UVA doses of 5 J/cm2 (UVA1) and 25 J/cm2 (UVA2). A total of 514 common proteins were detected for all three groups of cells. Moreover, more than half of these proteins changed their expression in response to UVA exposure even at low doses. Proteins associated with the biological process "Cell response to stress", (GO:0033554) were most strongly upregulated. 23 proteins associated with the biological process GO:0048870 - "cell motility", which are involved in the adaptive response to stress (e.g. UV radiation), were identified. 20 cell motility proteins were significantly upregulated, of which cytoskeletal actin-binding proteins were the major group. It is hypothesized that changes in the content of cell motility proteins in HaCaT keratinocytes can apparently be used to study physiological and pathological processes, particularly processes during wounding and wound healing.

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Immortalized HaCaT keratinocytes (from Bank of DKFZ, Heidelberg) before (control) and after exposure to ultraviolet A (5 J/cm2 and 25 J/cm2) during 48 h were used in the study. HaCaT keratinocytes were cultured in Petri dishes in DMEM medium supplemented with 10% FBS and antibiotics at 37°C in an atmosphere with 5% CO2. When confluency reached ~60-70%, keratinocytes were divided into 3 groups: control and two experimental groups (three Petri dishes for each group) and cultured for 48 hours. Then the cells were washed with DPBS solution and placed in the chamber of Vilber BIO-LINK dosed irradiation system (5 x 8-watt, 365 nm). The exposure time with the UVA source was selected so that the cumulative dose of absorbed radiation was 5 and 25 J/cm2. At the end of exposure, DPBS solution was replaced with culture medium and cells were incubated for 24 hours. Cells were detached using 0.25% trypsin-EDTA mixture (3 ml per Petri dish), centrifuged at 1200 rpm for 5 min, and then washed three times with ice-cold PBS. HaCaT cell pellet was placed into 500 μL of 0.2% SDS in 100 mM Tris-HCl (pH = 7.4), 120 mM NaCl, 5 mM EDTA, 1% PMSF and homogenized. After sonication (Sonopuls HD2070, “BANDELIN”), the samples were incubated for 30 min at + 4 °C on an orbital shaker. After heating at 95 °C for 4 min, they were centrifuged at 14,000 × g for 20 min (+ 4 °C). The lysate was collected, and the procedure was repeated starting from the sonication step. Lysates were pooled and centrifuged at 14,000 × g for 60 min (+ 4 °C); the finished supernatant was collected. The total protein concentration of HaCaT extracts was determined by the bicinchoninic acid assay on an Agilent 8453 UV–visible spectrophotometer with BSA as a standard. The 1DE-gel concentration protocol was then carried out to remove SDS. In-gel digestion with trypsin was performed according to the standard procedure. Separation and identification of the peptides were performed on an Ultimate 3000 nano-flow HPLC system (Dionex, USA), connected to a Orbitrap Q Exactive mass-spectrometer (Thermo Scientific, USA) equipped with a Nanospray Flex NG ion source (Thermo Scientific, USA) The obtained mass spectrometric data with the .raw extension were processed by the MaxQuant software (v. 1.6.3.4) with the built-in Andromeda search algorithm. Homo sapiens Swiss Prot/Uniprot complete proteome protein sequence database downloaded from the Uniprot database. The following parameters were used for subsequent processing: proteolytic cleavage enzyme was trypsin; the allowable error in measuring the monoisotopic mass of the peptide was ±0.01 Da, the allowable error in measuring the fragment ion was ±0.05 Da. Carbamide methylation of cysteine residues was chosen as a fixed chemical modification, and methionine oxidation was chosen as a variable modification.

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