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.