Effect of ultraviolet radiation on expression of transient receptor potential ankyrin 1 in HaCaT cells / 中华皮肤科杂志

Objective To investigate the photoregulation of transient receptor potential ankyrin 1 (TRPA1) in HaCaT cells,and to explore its mechanisms.Methods Cultured HaCaT cells were divided into 225-mJ/cm2 UVA radiation groups and 25-mJ/cm2 UVB radiation groups.HaCaT cells in the UVA radiation groups were further classified into 6 groups:blank control group 1 receiving no treatment,retinal group 1 treated with 12 μmol/L retinal alone,UVA group treated with 225 mJ/cm2 UVA radiation alone,retinal + UVA group (UVA-TRPA1 control group),retinal + UVA + 500 μmol/L cinnamaldehyde group (UVA-TRPA1 agonist group) and retinal + UVA + 1 mmol/L camphor group (UVA-TRPA1 antagonist group).Additionally,HaCaT cells in the UVB radiation groups were also further classified into 6 groups:blank control group 2 receiving no treatment,retinal group 2 treated with 12 μmol/L retinal alone,UVB group treated with 25-mJ/cm2 UVB radiation,retinal + UVB group (UVB-TRPA1 control group),retinal + UVB + 500 μmol/L cinnamaldehyde group (UVB-TRPA1 agonist group) and retinal + UVB + 1 mmol/L camphor group (UVB-TRPA1 antagonist group).Real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were performed to determine the mRNA and protein expression of TRPA 1 respectively.Flow cytometry was conducted to investigate changes of calcium influx in HaCaT cells in the above groups.Results qPCR and Western blot analysis showed that TRPA1 mRNA and protein were expressed in HaCaT cells.The fluorescence intensity of calcium influx significantly differed among the blank control group 1,retinal group 1,UVA group and retinal + UVA group (155.06 ± 7.62,148.37 ± 18.77,166.92 ± 3.71 and 331.333 ± 40.563;F =44.509,P ＜ 0.01),as well as among the blank control group 2,retinal group 2,UVB group and retinal + UVB group (150.20 ± 1.73,171.66 ± 56.23,147.56 ± 6.60 and 250.44 ± 9.13;F =85.261,P ＜ 0.01).Additionally,retinal + UVA/UVB groups showed significantly higher fluorescence intensity of calcium influx compared with the blank control groups (q =18.442,6.052,P ＜ 0.01).The TRPA1 agonist cinnamaldehyde and its antagonist camphor could regulate the UVA-and UVB-induced calcium influx (P ＜ 0.001).Compared with the blank control group 1 and 2 respectively,the fluorescence intensity of retinal-dependent calcium influx was significantly higher in the UVA/UVB-TRPA1 agonist group (q =14.934,32.770,P ＜ 0.001),and significantly lower in the UVA/UVB-TRPA1 antagonist group (q =7.986,14.596,P ＜ 0.001).Conclusion TRPA1 is expressed in HaCaT cells,and UVA or UVB can regulate the calcium influx in HaCaT cells by adjusting the activity of TRPA 1.

Effect of ultraviolet radiation on expression of transient receptor potential ankyrin 1 in HaCaT cells / 中华皮肤科杂志

Objective To investigate the photoregulation of transient receptor potential ankyrin 1 (TRPA1) in HaCaT cells,and to explore its mechanisms.Methods Cultured HaCaT cells were divided into 225-mJ/cm2 UVA radiation groups and 25-mJ/cm2 UVB radiation groups.HaCaT cells in the UVA radiation groups were further classified into 6 groups:blank control group 1 receiving no treatment,retinal group 1 treated with 12 μmol/L retinal alone,UVA group treated with 225 mJ/cm2 UVA radiation alone,retinal + UVA group (UVA-TRPA1 control group),retinal + UVA + 500 μmol/L cinnamaldehyde group (UVA-TRPA1 agonist group) and retinal + UVA + 1 mmol/L camphor group (UVA-TRPA1 antagonist group).Additionally,HaCaT cells in the UVB radiation groups were also further classified into 6 groups:blank control group 2 receiving no treatment,retinal group 2 treated with 12 μmol/L retinal alone,UVB group treated with 25-mJ/cm2 UVB radiation,retinal + UVB group (UVB-TRPA1 control group),retinal + UVB + 500 μmol/L cinnamaldehyde group (UVB-TRPA1 agonist group) and retinal + UVB + 1 mmol/L camphor group (UVB-TRPA1 antagonist group).Real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were performed to determine the mRNA and protein expression of TRPA 1 respectively.Flow cytometry was conducted to investigate changes of calcium influx in HaCaT cells in the above groups.Results qPCR and Western blot analysis showed that TRPA1 mRNA and protein were expressed in HaCaT cells.The fluorescence intensity of calcium influx significantly differed among the blank control group 1,retinal group 1,UVA group and retinal + UVA group (155.06 ± 7.62,148.37 ± 18.77,166.92 ± 3.71 and 331.333 ± 40.563;F =44.509,P ＜ 0.01),as well as among the blank control group 2,retinal group 2,UVB group and retinal + UVB group (150.20 ± 1.73,171.66 ± 56.23,147.56 ± 6.60 and 250.44 ± 9.13;F =85.261,P ＜ 0.01).Additionally,retinal + UVA/UVB groups showed significantly higher fluorescence intensity of calcium influx compared with the blank control groups (q =18.442,6.052,P ＜ 0.01).The TRPA1 agonist cinnamaldehyde and its antagonist camphor could regulate the UVA-and UVB-induced calcium influx (P ＜ 0.001).Compared with the blank control group 1 and 2 respectively,the fluorescence intensity of retinal-dependent calcium influx was significantly higher in the UVA/UVB-TRPA1 agonist group (q =14.934,32.770,P ＜ 0.001),and significantly lower in the UVA/UVB-TRPA1 antagonist group (q =7.986,14.596,P ＜ 0.001).Conclusion TRPA1 is expressed in HaCaT cells,and UVA or UVB can regulate the calcium influx in HaCaT cells by adjusting the activity of TRPA 1.

Abnormalities of erythrocyte membrane proteins in Korean patients with hereditary spherocytosis

Hereditary spherocytosis (HS) is a common inherited erythrocyte membrane disorder characterized by chronic hemolytic anemia. Clinical manifestations and biochemical abnormalities of HS are heterogeneous. In this study, we investigated erythrocyte membrane protein defects in 27 Korean HS cases. Utilizing both the Fairbanks system and the Laemmli system, sodium dodecyl sulfate polyacrylamide gel electrophoresis of erythrocyte membrane proteins was performed. Proteins were stained with Coomassie brilliant blue and gels were scanned using a densitometer. We detected spectrin deficiency in 7.4% of cases (2/27), ankyrin deficiency in 29.6% (8/27), combined spectrin and ankyrin deficiency in 3.7% (1/27), band 3 deficiency in 11.1% (3/27) and protein 4.2 deficiency in 14.8% (4/27). Membrane protein deficiencies were not observed in nine cases (33.3%, 9/27). Members of two of seven families tested showed the same protein defects as the proband. Ankyrin deficiency alone and combined with spectrin deficiency accounted for 33.3% of cases (9/27), and they were the most common biochemical defects in Korean HS cases. Protein 4.2 deficiency caused HS more frequently in Koreans than in Caucasians.

Abnormalities of erythrocyte membrane proteins in Korean patients with hereditary spherocytosis

Hereditary spherocytosis (HS) is a common inherited erythrocyte membrane disorder characterized by chronic hemolytic anemia. Clinical manifestations and biochemical abnormalities of HS are heterogeneous. In this study, we investigated erythrocyte membrane protein defects in 27 Korean HS cases. Utilizing both the Fairbanks system and the Laemmli system, sodium dodecyl sulfate polyacrylamide gel electrophoresis of erythrocyte membrane proteins was performed. Proteins were stained with Coomassie brilliant blue and gels were scanned using a densitometer. We detected spectrin deficiency in 7.4% of cases (2/27), ankyrin deficiency in 29.6% (8/27), combined spectrin and ankyrin deficiency in 3.7% (1/27), band 3 deficiency in 11.1% (3/27) and protein 4.2 deficiency in 14.8% (4/27). Membrane protein deficiencies were not observed in nine cases (33.3%, 9/27). Members of two of seven families tested showed the same protein defects as the proband. Ankyrin deficiency alone and combined with spectrin deficiency accounted for 33.3% of cases (9/27), and they were the most common biochemical defects in Korean HS cases. Protein 4.2 deficiency caused HS more frequently in Koreans than in Caucasians.