Thymosin beta 4 suppression of corneal NFkappaB: a potential anti-inflammatory pathway.
The purpose of this study was to determine the effect of thymosin beta 4 (Tbeta4) on NFkappaB protein levels, activation, phosphorylation, and nuclear translocation in a model of tumor necrosis factor (TNF)-alpha-mediated corneal inflammation. Transformed and primary (HCET and HCEC) human corneal epithelial cells were stimulated with the pro-inflammatory cytokine TNF-alpha and treated or not with Tbeta4. Nuclear NFkappaB p65 subunit protein levels were assayed using ELISA, and activity was measured by determining NFkappaB binding to consensus oligonucleotides. NFkappaB p65 protein phosphorylation was also measured by ELISA. Nuclear translocation of NFkappaB p65 subunit was assayed by immunofluorescence microscopy. Compared to non-treated controls, Tbeta4 treatment significantly decreased nuclear NFkappaB protein levels, NFkappaB activity and p65 subunit phosphorylation in corneal epithelial cells after TNF-alpha stimulation. In TNF-alpha-stimulated corneal epithelial cells, NFkappaB p65 subunit translocation to the nucleus was observed using immunofluorescence microscopy. In contrast, Tbeta4 blocked nuclear translocation of the NFkappaB p65 subunit in TNF-alpha-stimulated corneal epithelial cells. TNF-alpha initiates cell signaling pathways that converge on the activation of NFkappaB, thus both are known mediators of the inflammatory process. Tbeta4, a protein with diverse cellular functions including wound healing and suppression of inflammation, inhibits the activation of NFkappaB in TNF-alpha-stimulated cells. These results have important clinical implications for the potential role of Tbeta4 as a corneal anti-inflammatory agent.
Sosne
Gabriel
G
Department of Ophthalmology, Kresge Eye Institute, Wayne State University School of Medicine, 540 E. Canfield, Scott Hall 8314, Detroit, MI 48201, USA. gsosne@med.wayne.edu
Qiu
Ping
P
Christopherson
Patricia L
PL
Wheater
Michelle Kurpakus
MK
D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007634:Keratitis / Q000276:immunology / Q000378:metabolism*; D008840:Microfilament Proteins / Q000494:pharmacology*; D008856:Microscopy, Fluorescence / Q000379:methods; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D013947:Thymosin / Q000494:pharmacology*; D051996:Transcription Factor RelA / Q000032:analysis; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology; D000099201:Thymosin beta(4)
D005136:Eye Proteins; D008840:Microfilament Proteins; D016328:NF-kappa B; D013947:Thymosin; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000099201:Thymosin beta(4)