Clathrin heavy chain is required for TNF-induced inflammatory signaling.
BACKGROUND
Tumor necrosis factor receptor I recruits tumor necrosis factor receptor-associated death domain (TRADD) and multiple kinases that ultimately phosphorylate inhibitor kappa B (IKB alpha). Degradation of phospho-IKB alpha (p-IKB alpha) frees nuclear factor kappa B (NFKB) to be active and phosphorylated. Many receptors require clathrin-mediated endocytosis to provide the scaffolds necessary for signaling. Therefore, we investigated the role of clathrin heavy chain (CHC) in tumor necrosis factor alpha (TNF-alpha)-induced IKB alpha phosphorylation and NFKB activation. We hypothesized that CHC was required for TNF-alpha-induced inflammatory signaling.


METHODS
We treated human pulmonary epithelial cells with small interfering RNA to knock down intracellular CHC (CHCsil). TRADD and scrambled (noncoding) small interfering RNA sequences were used as positive and negative controls, respectively. Treatment groups were exposed to 10 ng/mL of TNF-alpha. Total I kappaB alpha, p-I kappaB alpha, and phosphorylated P65 (a subunit of NFKB) were determined by immunoblot staining. Densitometry was normalized to controls for the analysis of the stains. TNF-alpha-induced release of monocyte chemoattractant protein 1 (MCP-1) was determined by enzyme-linked immunosorbent assay. Statistical analyses were determined by analysis of variance or paired t test as appropriate.


RESULTS
TNF-alpha-induced I kappaB alpha phosphorylation and degradation at 5 and 30 minutes, respectively, and induced P65 phosphorylation. CHCsil diminished p-I kappaB alpha by 91% (P < .03); however, I kappaB alpha degradation was not affected. CHC knockdown caused a 66% decrease in P65 phosphorylation after 3 minutes of TNF-alpha. CHCsil decreased TNF-alpha-induced MCP-1 by 46% (P < .05), compared with control.


CONCLUSIONS
CHCsil significantly impairs phosphorylation of both I kappaB alpha and P65. CHCsil also significantly decreased MCP-1 production. These data suggest that CHC is required for certain TNF-alpha-induced, inflammatory signaling pathways.
Escobar
Guillermo A
GA
University of Colorado Health Sciences Center, Denver, CO 80262, USA.
McIntyre
Robert C
RC
Moore
Ernest E
EE
Gamboni-Robertson
Fabia
F
Banerjee
Anirban
A
D018929:Cell Culture Techniques; D018932:Chemokine CCL2 / Q000378:metabolism; D033922:Clathrin Heavy Chains / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation; D034741:RNA, Small Interfering; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D051996:Transcription Factor RelA / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D018932:Chemokine CCL2; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D033922:Clathrin Heavy Chains; D000072000:NF-KappaB Inhibitor alpha