Despite athe critical importance of dietary iron absorption in maintaining iron balance, our understanding of the molecular events involved in the uptake and transport of this essential nutrient across the intestinal mucosa remains limited. This pilot study proposes new approaches in our efforts to understand the molecular basis of intestinal iron absorption through immunological analysis. Thus, the feasibility/pilot study will potentially produce the necessary reagents for the molecular cloning of factors involved in the transport process and the identification of regulatory elements involved in modulating iron assimilation in the gut. Recent advances in our understanding of the transcriptional regulation by NFE2 of factors involved in maintaining iron balance, and specifically those involved in erythroid/intestinal iron transport, will enable us to achieve our goals. It has now been established that mk/mk mice lack functional NF-E2, resulting in defective intestinal iron absorption, while beta-thalassemic mice (Hbbd.th31Hbbd.th3) exhibit enhanced iron absorption associated with increased levels NF-E2. Therefore, these two mutants can be utilized to identify proteins lacking in the homozygous mk mouse that are regulated by NF-E2 and potentially involved in intestinal iron transport. Our goals are; 1.) to raise a panel of rat anti-mouse monoclonal antibodies that recognize proteins of microvillous membranes of beta-thalassemic intestine and 2.) to screen antibodies against intestinal membrane preparations from normal (DBA/2J) and mk/mk mice in order to specifically identify those monoclonal lines that recognize proteins present int he beta-thalassemic preparation but lacking in the mk mutants. These factors may be essential for iron uptake as well as other facets of iron metabolism subject too regulatory control by NF-E2. Since the beta-thalassemic mice express greatly increased levels of NF-E2, it is anticipated that the panel of antibodies will broadly recognize proteins that are transcriptionally regulated by this factor and that are intimately tied to the maintenance of iron balance. Using selected hybridomas, our final goals is: 3.) To test the ability of monoclonal antibodies to inhibit iron uptake and to immunoprecipitate ferrireductase and/or iron-binding activities. Our laboratory has established assays that have characterized each of these individual elements in iron transport which minimally involves three functions;: an iron-binding component, a ferrireductase activity and a Fe-specific channel or carrier. For this analysis, three model systems will be employed: everted duodenal loops from normal, beta-thalassemic and mk/mk mice; rat IEC-6 and/or the human Caco-2 intestinal cell lines; and human K562 erythroleukemia cells (known to express NF-E2). This investigation will provide for the identification of those antigens involved in iron uptake and assimilation and will initiate future studies leading to the ultimate molecular cloning of factors that mediate intestinal iron absorption and the identification of factors regulating this process.
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