Effects of the 24 N-terminal Amino Acids of p55PIK on Endotoxinstimulated Release of Inflammatory Cytokines by HaCaT Cells

• Published in: Journal of Huazhong University of Science and Technology. Medical sciences 2013-08, Vol.33 (4), p.587-593
• Main Authors: Lv, Feng, You, Wei, Yu, Yang, Hu, Jun-bo, Zhang, Bin, Wang, Jing
• Format: Article
• Language: English
• Subjects: Amino Acids - metabolism; Cell Line; Cytokines - metabolism; Endotoxins - metabolism; HaCaT细胞; Humans; Inflammation - metabolism; Medicine; Medicine & Public Health; N-端氨基酸; NF-kB; Phosphatidylinositol 3-Kinases - metabolism; TNF-α; Western印迹法; 共聚焦激光扫描显微镜; 炎性细胞因子; 磷脂酰肌醇3激酶
• ISSN: 1672-0733; 1993-1352
• DOI: 10.1007/s11596-013-1163-2

Summary： This study aimed to examine the effect of the 24 N-terminal amino acids （N24） ofp55PIK, a regulatory subunit of phosphatidylinositol 3-kinase （PI3K）, on the endotoxin lipopolysaccharide （LPS）-stimulated release of the cytokines （CKs） by HaCaT cells. The fusion protein, trans-acting activator of transcription （TAT）-N24 （an experimental peptide, EP） containing the N24 of PI3K-p55PIK, was constructed, and TAT-N24 fusion peptide was expressed and identified in BL21 E.coli. HaCaT cells （a human keratinocyte cell line） was cultured and stimulated by LPS at 100 ng/mL for 1, 2, 4, 8, 16 or 24 h, or by LPS at 10, 100 ng/mL, 1, 10 or 100 μg/mL of for 4 h. Changes in the protein and mRNA levels of tumor necrosis factor-alpha （TNF-ct）, interleukin-6 （IL-6） and interleukin-8 （IL-8） released by HaCaT cells following EP intervention were determined by enzyme-linked immunosorbent assay （ELISA） and real-time polymerase chain reaction （PCR）. Immunofluorescence confocal laser scanning microscopy was utilized to detect the protein expression and translocation of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells （NF-r,B p65） in HaCaT cells. The expression of the NF-kB inhibitor alpha （Iv, B-a） protein in LPS-stimulated HaCaT cells after the EP intervention was measured by Western blotting. The resillts showed that EP treatment increased TNF-a secretion from HaCaT cells. EP at certain concentrations could effectively inhibit the LPS-stimulated release of TNF-a, IL-6 and IL-8 from HaCaT cells. The ELISA assay demonstrated that the concentrations of TNF-a, IL-6 and IL-8 in the supernatants of LPS-stimulated cells were reduced from 208.06±30.18, 86.4±9.78 and 260.59±54.05 pg/mL to 121.78±22.26, 53.18±7.36 and 125.08±35.17 pg/mL, respectively, in the supernatants of cells treated by LPS and EP combined. Real-time PCR also revealed that the expression of the three pro-inflammatory CKs was significantly decreased after EP intervention. Immunofluorescence confocal laser scanning microscopy showed that NF-kB p65 protein was primarily expressed in the cytoplasm of non-stimulated HaCaT cells. After LPS stimulation, NF-kB p65 was translocated into the nucleus, and the nuclear expression of this protein increased. The nuclear NF-kB p65 protein expression was inhibited after the addition of EP. Western blotting showed that Ir, B-a expression began to decrease 30 min after LPS stimulation and declined to a trough 4 h later. Ir, B-a expression began to