| Abstract | OBJECTIVE: The zinc-finger protein tristetraprolin (TTP) has been demonstrated to regulate tumor necrosis factor alpha (TNFalpha) messenger RNA (mRNA) instability in murine macrophages. We sought to develop a model system to characterize the effects of human TTP (hTTP) on TNFalpha 3'-untranslated region (3'-UTR)-mediated expression. We also generated a specific polyclonal antibody against hTTP that enabled the examination of the subcellular distribution of hTTP and its RNA binding in vivo. METHODS: Transfection of reporter gene constructs were used to functionally characterize the role of hTTP in regulating TNFalpha expression in a 3'-UTR-dependent manner. An immunoprecipitation reverse transcription-polymerase chain reaction technique, immunoblotting, immunocytochemistry, and sucrose density fractionation were used to identify and localize hTTP. RESULTS: We found that hTTP interacted with human TNFalpha mRNA in the cytoplasm. The presence of the TNFalpha 3'-UTR was sufficient to confer binding by TTP in vivo. This interaction resulted in reduced luciferase reporter gene activity in a TNFalpha 3'-UTR adenine-uridine-rich element (ARE)-dependent manner. Immunoblotting and immunocytochemistry indicated that endogenous and transfected hTTP localized to the cytoplasm. Results of sucrose density fractionation studies were consistent with a polysomal location of hTTP. In rheumatoid synovium, hTTP expression was restricted to cells in the synovial lining layers. CONCLUSION: Through the development of an antiserum specific for hTTP, we have been able to demonstrate that hTTP binds specifically to the TNFalpha 3'-UTR and reduces reporter gene expression in an ARE-specific manner. These studies establish that hTTP is likely to function in a similar, if not identical manner, in the posttranscriptional regulation of TNFalpha. Understanding the posttranscriptional regulation of TNFalpha biosynthesis is important for the development of novel treatment strategies in rheumatoid arthritis. |
| Authors | Seth A Brooks, John E Connolly, Roger J Diegel, Roy A Fava, William F C Rigby
(Affiliation: Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire, USA.)
|
| Journal | Arthritis and rheumatism
(Arthritis Rheum)
Vol. 46
Issue 5
Pg. 1362-70
(May 2002)
ISSN: 0004-3591 United States |
| PMID | 12115244
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)
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| Chemical References |
- 3' Untranslated Regions
- DNA-Binding Proteins
- Immediate-Early Proteins
- RNA, Messenger
- Tristetraprolin
- Tumor Necrosis Factor-alpha
- ZFP36 protein, human
- Luciferases
|
| Topics |
- 3' Untranslated Regions
(physiology)
- Antibody Specificity
- Arthritis, Rheumatoid
(immunology)
- Base Sequence
- Cell Line
- DNA-Binding Proteins
- Gene Expression
(physiology)
- Humans
- Immediate-Early Proteins
(analysis, genetics, immunology)
- Kidney
(cytology)
- Luciferases
(genetics)
- Molecular Sequence Data
- Myeloid Cells
(chemistry)
- RNA Processing, Post-Transcriptional
(physiology)
- RNA, Messenger
(analysis)
- Transfection
- Tristetraprolin
- Tumor Necrosis Factor-alpha
(genetics, immunology)
|
