title: Mechanisms by which the N-terminal 24 amino acids of the p55 regulatory subunit of phosphatidylinositol 3-kinase affect endotoxin-induced cytokine release in human keratinocytes.
abstract: To understand the association between the cytokine network and psoriasis, the present study cultured human keratinocytes (HaCaT cells) and investigated the effects of the phosphatidylinositol‑3‑kinase (PI3K) p55 regulatory subunit (p55PIK), and its N‑terminal 24 amino acids (N24) on the regulation of endotoxin (LPS)‑induced cytokine secretion. The results of the enzyme‑linked immunosorbent assay and reverse transcription quantitative polymerase chain reaction revealed an increased release of the inflammatory cytokines, tumor necrosis factor (TNF)‑α, interleukin (IL)‑6 and IL‑8 in the HaCaT cells following LPS stimulation. Transfection with the adenovirus (AD)‑N24‑green fluorescent protein (GFP) suppressed the release of these cytokines, whereas AD‑p55PIK‑GFP increased their release. Immunocytochemistry detected a low level of nuclear factor (NF)‑κB p65 staining in quiescent HaCaT cells, which was localized primarily in the cytoplasm. LPS stimulation induced the translocation of NF‑κB p65 protein into the nucleus and intense staining suggested increased expression. Transfection with AD‑N24‑GFP reduced the expression of NF‑κB p65 in the nucleus. Western blot analysis demonstrated that AD‑N24‑GFP downregulated the expression levels of the Toll‑like receptor (TLR)2/TLR4/myeloid differentiation factor 88 (MyD88) pathway components in the HaCaT cells, without affecting the PI3K/Akt signaling pathway. Transfection with AD‑p55PIK‑GFP resulted in an increased expression level of MyD88 protein and phosphorylated Akt. Co‑transfection with AD‑N24‑GFP and AD‑p55PIK‑GFP did not significantly alter the levels of phosphorylated extracellular‑signal‑regulated kinases 1/2, c‑Jun N‑terminal kinases or p38, indicating that AD‑N24‑GFP and AD‑p55PIK‑GFP did not affect the mitogen‑activated protein kinase signaling pathway. In conclusion, AD‑N24‑GFP effectively inhibited the LPS‑induced expression levels of TNF‑α, IL‑6 and IL‑8. The elevated expression of p55PIK synergized with LPS and promoted the release of inflammatory cytokines. AD‑N24‑GFP and AD‑p55PIK‑GFP affected LPS‑induced inflammatory cytokine release in the HaCaT cells through the TLRs/MyD88 signaling pathways.
authors.affiliation: Department of Breast Surgery, Henan Provincial People's Hospital, The People's Hospital of Zhengzhou University, Zhengzhou, Henan 450003, P.R. China.
authors.forename: Feng
authors.initials: F
authors.lastname: Lv
authors.affiliation: Department of Breast Surgery, Henan Provincial People's Hospital, The People's Hospital of Zhengzhou University, Zhengzhou, Henan 450003, P.R. China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Department of Molecular Medical Center, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hebei 430030, P.R. China.
authors.forename: Guihua
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Molecular Medical Center, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hebei 430030, P.R. China.
authors.forename: Junbo
authors.initials: J
authors.lastname: Hu
authors.affiliation: Department of Breast Surgery, Henan Provincial People's Hospital, The People's Hospital of Zhengzhou University, Zhengzhou, Henan 450003, P.R. China.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Breast Surgery, Henan Provincial People's Hospital, The People's Hospital of Zhengzhou University, Zhengzhou, Henan 450003, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: You
authors.affiliation: Department of Immunology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hebei 430030, P.R. China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
mesh_terms: D015415:Biomarkers; D045744:Cell Line, Tumor; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D004731:Endotoxins / Q000494:pharmacology*; D020869:Gene Expression Profiling; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D015415:Biomarkers; D016207:Cytokines; D004731:Endotoxins; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D019869:Phosphatidylinositol 3-Kinases; C523442:PIK3R3 protein, human