The long-term goal of these studies is to define the biochemical, cellular and molecular mechanisms by which inflammatory stimuli lead to the acute phase response. This phenomenon, observed in humans in the first days following bacterial infections, trauma and tissue infarction, and in a variety of neoplasias and chronic inflammatory states, appears to represent a primary defense mechanism prior to the immune response. Among the changes seem in the acute phase response is a rapid and dramatic increase in hepatic synthesis of two plasma proteins, C-reactive protein (CRP) and serum amyloid A (SAA). Induction of SAA in the human hepatoma cell line Hep 3B requires the synergistic interaction of the cytokines interleukin 6 (IL-6) and either interleukin I (IL-1) or tumor necrosis factor alpha (TNF), while CRP induction requires--,he combination of IL-6 plus IL-1. Dexamethasone (dex) potentiates induction of both proteins by cytokines. In addition, another cytokine, transforming growth factor beta-1 (TGF-beta) has been found to inhibit SAA and CRP induction. Recent studies suggest that induction of SAA and CRP involves both transcriptional and post-transcriptional regulation, and that IL-1 and TGFbeta may exert their effects on CRP at the translational level.
The specific aims of these studies are to make use of the Hep 3B cell line model to determine the extent to which regulation at the levels of transcription, mRNA stability and translational efficiency are involved in regulation of SAA and CRP biosynthesis by the synergistic combinations of cytokines and dex described above. Initial studies will delineate the kinetics of transcription, mRNA accumulation and synthesis of SAA and CRP following exposure to inducing agents, employing nuclear run-on, Northern blot and metabolic labelling techniques. If those studies suggest post-transcriptional or translational regulation, as expected, further studies will directly evaluate effects of cytokines and dex on specific mRNA stability, employing inhibitors of transcription, and on translational efficiency, estimated by assessing ribosomal association with specific mRNA. Finally, the effects of inhibitors of protein synthesis on mRNA stability and accumulation will be assessed. These studies will cast light on the mechanisms regulating the body's primary defense mechanisms against infection and tissue injury, and are especially relevant to the elderly, who are particularly vulnerable to these processes.
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