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

2015 Molecular Medicine Reports  
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
more » ... 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.
doi:10.3892/mmr.2015.3184 pmid:25585688 fatcat:5kl2ftwugbgmzd4cbzelvino4m