Neonatal jaundice is a condition that effects children throughout the world. Pathologic jaundice becomes a serious threat to the well-being of neonates in the context of hemolytic disease which is associated with increased bilirubin production. Our laboratory's approach to this problem is to monitor bilirubin production, and assess the efficacy of therapeutic agents that inhibit the activity of key enzymes, heme oxygenase isoenzymes 1 and 2 (HO-1,2), in the catalyses of heme to bilirubin. Expression of the HO-1 isoenzyme varies both in culture and in vivo in response to metabolic cues such as changes in heme concentrations. Metalloporphyrins, heme analogs, are clinically relevant inhibitors of HO enzymatic activity, but some have also been shown to increase HO-1 expression at the level of transcription which may offset therapeutic uses. Here we propose to address the regulation of HO transcription in culture and living animals in the presence and absence of enzyme inhibitors. This proposal contains three specific aims. First, full length, truncated, and condensed HO-1 promoters will be placed upstream of the luciferase coding sequence, and stable cell lines will be generated from this set of promoter-reporter gene fusions. The activity of the HO-1 promoter will be assessed in culture and compared to previously described patterns of expression to map regions of this promoter that respond to specific stimuli. Second, a selected series of metalloporphyrins with potential clinical use will be tested for effects on HO-1 transcription and enzyme activity in culture. Lastly, using our recently developed in vivo monitoring method, modulators of HO gene expression will be evaluated in living animals to determine the in vivo effects of inhibitors of HO enzymatic activity in real time. Differential regulation of both HO isoenzymes will be compared, first in culture and then in living animals, using luciferase reporters with two different colors of emission. An inability to correlate in vitro or cell culture data with in vivo observations is a familiar problem in biological chemistry. Here we attempt to address this issue using an integrated approach where the same set of reporter constructs is used in vitro and in vivo, and similarly monitored in both environments. As such, this proposal describes analyses of HO expression that will allow more precise hypotheses about gene expression and specific pathway inhibition. We can then combine this data without knowledge of pharmacologic properties of HO inhibitors to select compounds that control hyperbilirubinemia and prevent or treat neonatal jaundice.
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